Smac/diablo inhibitors useful for treating cancer

ABSTRACT

The present invention relates to compositions and methods for treating cancer, particularly to agents that inhibit the expression and/or activity of the protein second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (SMAC/Diablo). The inhibiting agents include RNA interference molecules silencing the expression of SMAC/Diablo and peptides modulating its interactions within the cell nucleus and mitochondria. The methods and agents of the present invention are useful in treating cancers associated with overexpression of SMAC/Diablo.

The Sequence Listing in ASCII text file format of 81,175 bytes in size,created on May 10, 2021, with the file name“2021-05-10SequenceListing_SHOSHAN11,” filed in the U.S. Patent andTrademark Office on even date herewith, is hereby incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for treatingcancer, particularly to agents that inhibit the expression and/oractivity of the protein second mitochondria-derived activator ofcaspase/direct inhibitor of apoptosis-binding protein with low pI(SMAC/Diablo), including RNA interference molecules silencing theexpression of SMAC/Diablo and peptides modulating its activity, usefulfor treating cancers associated with overexpression of SMAC/Diablo.

BACKGROUND OF THE INVENTION

The canonical SMAC/Diablo (second mitochondria-derived activator ofcaspase/direct inhibitor of apoptosis-binding protein with low pI)isoform, SMAC-α, is a mitochondrial intermembrane space (IMS)pro-apoptotic protein (Verhagen, A. M., et al. 2000. Cell 102, 43-53;Du, Cet al. 2000. Cell 102, 33-42). The N-terminus of SMAC/Diablo servesas a mitochondrial targeting signal (MTS), and is cleaved to form themature 26 kDa protein. Following the induction of apoptosis, SMAC/Diablois released into the cytosol (Du, Cet al. 2000, ibid; Deng, Y., et al.2002. Genes Dev. 16, 33-45), where it interacts with members of theprotein family designated “inhibitor of apoptosis proteins” (IAPs),including cIAP1, cIAP2, and XIAP to neutralize the inhibitory effects ofIAPB on caspases and, thus, initiates apoptosis (Shiozaki, E. N., andShi, Y. 200). Trends Biochem. Sci. 29, 486-494; Verhagen, A. M., andVaux, D. L. 2002. Apoptosis 7, 163-166). This interaction neutralizesthe inhibitory effects of IAPB on caspases, and thus initiatesapoptosis. In interacting with IAPB, SMAC/Diablo acts as a homodimer,mediated via an N-terminal motif (Ala-Val-Pro-Ile). In addition,SMAC/Diablo was shown to be controlled by several other proteinsincluding the Bcl-2 family of proteins, mitogen-activated protein kinasefamily members, (e.g. Erk1/2) and c-Jun N-terminal kinase.

Although there are number of SMAC/Diablo variants generated byalternative splicing, SMAC/Diablo-α is the main IAP inhibitor (Adrain,C., et al. 2001. EMBO J 20, 6627-6636). Another isoform, SMAC/Diablo-β(Also known as SMAC-S), which lacks both the IAP-binding motif (IBM) andthe MTS, can sensitize cells to apoptosis when over-expressed,suggesting that SMAC/Diablo may also serve functions that are IBM- andmitochondria-independent. A cytosolic form, SMAC/Diablo-ε, which alsolacks both IBM and MTS elements, is ubiquitously expressed in normalhuman tissues and cancer cell lines (Martinez-Ruiz, G. U., et al., 2014.Int J Clin Exp Pathol 7, 5515-5526), is not involved in apoptosis andhas been shown to be associated with tumorigenicity (Grimshaw, M. J., etal., 2008 Breast Cancer Res 10, R52). Additional isoforms of SMAC/Diabloare SMAC/Diablo-δ (also known and SMAC/Diablo-3), SMAC/Diablo-4 andSMAC/Diablo-γ (also known as SMAC/Diablo-5).

Mice lacking SMAC/Diablo are viable, grow and mature normally, presentembryonic fibroblasts, lymphocytes, and hepatocytes without anyhistological abnormalities, and exhibit wild-type responses to all typesof apoptotic stimuli (Okada, H., et al., 2002 Mol Cell Biol 22,3509-3517).

SMAC/Diablo was found to be down-regulated in certain types of cancers.For instance, the expression levels of both SMAC/Diablo mRNA and proteinwere reduced in hepatocellular carcinoma cells, as compared to normalhepatic tissue (Bao, S. T., et al., 2006. Hepatobiliary Pancreat Dis Int5, 580-583). Over-expression of recombinant SMAC/Diablo was found tosensitize neoplastic cells to apoptotic death (Kashkar, H., et al.,2006. Blood 108, 3434-3440). SMAC/Diablo or mimetic molecules thereofhave been proposed as anti-cancer agents capable of inhibiting IAPB andthus promoting cancer cell death (for example, U.S. ApplicationPublication Nos. 2013/0196927; U.S. Pat. Nos. 7,816,538, 7,884,211, and9,861,679).

U.S. Patent Application Publication No. 2008/0253966 discloses methodsfor diagnosing and providing prognosis of cancer diseases thatunderexpress SMAC/Diablo, and methods for treating or inhibiting suchdiseases.

Unexpectedly, despite its role in promoting cell death, SMAC/Diablo wasfound to be over-expressed in some cancers and higher levels of bothSMAC/Diablo protein and its encoding mRNA were reported in cervicalcancer (Arellano-Llamas, A., et al., 2006. BMC Cancer 6, 256), lung,ovarian and prostate carcinomas, different types of sarcoma (Yoo, N. J.,et al., 2003. APMIS 111, 382-388), gastric carcinomas (Shintani, M., etal., 2014. Oncol Lett 8, 2581-2586), pancreatic cancer (Hu H. Y. et al.,2012. Hepatogastroenterology 59(120):2640-3), testis cancer (Yoo N. J.et al., 2003. APMIS 111(3):382-388) and renal cell carcinoma(Kempkensteffen, C., et al., 2008. J Cancer Res Clin Oncol 134,543-550).

There is a growing awareness of the understanding that cancer is not asingle disease. Thus, there is a need for and it would be highlyadvantageous to have compositions and methods for treating cancerdiseases that share common characteristics.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for treatingcancers that overexpress SMAC/Diablo. The present invention providesagents that inhibit the expression and/or activity of SMAC/Diablo. Inparticular embodiments the present invention provides RNA inhibitoryagents that silence or otherwise down regulate the expression ofSMAC/Diablo and peptides that interact with SMAC/Diablo protein withinthe mitochondria and/or nucleus.

The present invention is based in part on the unexpected discovery thatsilencing the expression of SMAC/Diablo encoding mRNA, using smallinterfering RNA (siRNA) molecules, in cancer cell lines overexpressingthe mRNA and encoded protein, markedly reduced the cell proliferation.Silencing SMAC/Diablo in mice subcutaneous xenografts of lung cancercells or of breast triple negative cancer cells significantly reducedtumor growth. Furthermore, following SMAC/Diablo silencing treatment,residual lung tumors demonstrated morphological changes, including thedevelopment of alveoli-like anatomical structures and elimination ofintracellular organelles, such as lamellar bodies, which are typical toalveolar type 2 (AT2) cells in lung tissue and non-small cells lungcarcinoma cells. Next-generation sequencing of mRNA material obtainedfrom tumors treated with SMAC-silencing siRNA molecules revealed alteredexpression of genes associated with intercellular membranal and exosomalnetworks, cells differentiation, lipid metabolism and transportactivities. SMAC/Diablo silencing in lung cancer tumors decreased thelevel of phospholipids, including phosphatidylcholine, and theexpression of enzymes associated with their synthesis. The presentinvention further discloses peptides derived from proteins interactingwith SMAC/Diablo and modulate its activity. The peptides specificallybind to SMAC/Diablo and, when targeted into the mitochondria and/ornucleus of cancerous cells overexpressing SMAC/Diablo, inhibit the cellproliferation.

According to one aspect, the present invention provides a method fortreating cancer associated with over-expression of secondmitochondria-derived activator of caspase/direct inhibitor ofapoptosis-binding protein with low pI (SMAC/Diablo) in a subject in needthereof, comprising administering to the subject a therapeuticallyeffective amount of at least one agent that inhibits the expressionand/or activity of the SMAC/Diablo.

According to certain exemplary embodiments, inhibiting the expressionand/or activity of SMAC/Diablo results in reduced proliferation ofcancerous cells.

According to certain embodiments, the agent that inhibits the expressionand/or activity of SMAC/Diablo is selected from the group consisting ofan inhibitory nucleic acid, an inhibitory peptide, an inhibitory smallmolecule, an inhibitory aptamer, and combinations thereof.

According to certain embodiments, the agent is SMAC/Diablo silencingoligonucleotide or a recombinant construct encoding same, targeted tothe gene or mRNA sequence encoding SMAC/Diablo protein.

According to certain embodiments, the SMAC/Diablo encoding genecomprises a nucleic acid sequence at least 80%, at least 85%, at least90%, at least 95% or more homologous to the nucleic acid sequence setforth in SEQ ID NO:1 (NG_029459.1).

According to certain embodiments, the SMAC/Diablo protein is selectedfrom the group consisting of SMAC/Diablo-α, having an amino acidssequence at least 80%, at least 85%, at least 90% or more homologous tothe amino acid sequence set forth in SEQ ID NO:2; SMAC/Diablo-δ, havingan amino acids sequence at least 80%, at least 85%, at least 90% or morehomologous to the amino acid sequence set forth in SEQ ID NO:4;SMAC/Diablo-β, having an amino acids sequence at least 80%, at least85%, at least 90% or more homologous to the amino acid sequence setforth in SEQ ID NO:6; SMAC/Diablo-4, having an amino acids sequence atleast 80%, at least 85%, at least 90% or more homologous to the aminoacid sequence set forth in SEQ ID NO:8; and SMAC/Diablo-γ, having anamino acids sequence at least 80%, at least 85%, at least 90% or morehomologous to the amino acid sequence set forth in SEQ ID NO:10. Eachpossibility represents a separate embodiment of the present invention.

According to certain embodiments, the SMAC/Diablo-α is encoded by anucleic acid sequence at least 80%, at least 85%, at least 90% or morehomologous to the nucleic acid sequence set forth in SEQ ID NO:3(NM_019887.5); the SMAC/Diablo-δ is encoded by a nucleic acid sequenceat least 80%, at least 85%, at least 90% or more homologous to thenucleic acid sequence set forth in SEQ ID NO:5 (NM_001278342.1); theSMAC/Diablo-β is encoded by a nucleic acid sequence at least 80%, atleast 85%, at least 90% or more homologous to the nucleic acid sequenceset forth in SEQ ID NO:7 (NM_001278304.1; NM_138930.3); theSMAC/Diablo-4 is encoded by a nucleic acid sequence at least 80%, atleast 85%, at least 90% or more homologous to the nucleic acid sequenceset forth in SEQ ID NO:9 (NM_001278302.1); and the SMAC/Diablo-γ isencoded by a nucleic acid sequence at least 80%, at least 85%, at least90% or more homologous to the nucleic acid sequence set forth in SEQ IDNO:11 (NM_001278303.1). Each possibility represents a separateembodiment of the present invention.

According to certain embodiments, the SMAC/Diablo protein is humanprotein (hSMAC/Diablo)selected from the group consisting ofhSMAC/Diablo-α, having the amino acid sequence set forth in SEQ ID NO:2;hSMAC/Diablo-δ, having the amino acid sequence set forth in SEQ ID NO:4;hSMAC/Diablo-δ, having the amino acid sequence set forth in SEQ ID NO:6;hSMAC/Diablo-4, having the amino acid sequence set forth in SEQ ID NO:8;and hSMAC/Diablo-γ, having the amino acid sequence set forth in SEQ IDNO:10. Each possibility represents a separate embodiment of the presentinvention.

According to certain embodiments, the hSMAC/Diablo-α is encoded by thenucleic acid sequence having SEQ ID NO:3; hSMAC/Diablo-δ is encoded bythe nucleic acid sequence having SEQ ID NO:5; hSMAC/Diablo-β is encodedby the nucleic acid sequence having SEQ ID NO:7; hSMAC/Diablo-4 isencoded by the nucleic acid sequence having SEQ ID NO:9; andhSMAC/Diablo-γ is encoded by the nucleic acid sequence having SEQ IDNO:11. Each possibility represents a separate embodiment of the presentinvention.

According to some embodiments, the SMAC/Diablo protein is selected fromthe group consisting of SMAC/Diablo-α and SMAC/Diablo-δ.

According to certain embodiments, the SMAC/Diablo silencing moleculecomprises at least 15 consecutive nucleic acid bases targeted to(hybridizable with) a nucleic acid sequence encoding SMAC/Diablo proteinor to a complementary polynucleotide thereof.

According to certain embodiments, the SMAC/Diablo silencing moleculecomprises at least 15 consecutive nucleic acid bases substantiallyidentical to a nucleic acid sequence at least 80%, at least 85%, atleast 90% or more homologous to the nucleic acid sequence set forth inSEQ ID NO:3 or to a nucleic acid sequence complementary to SEQ ID NO:3.

According to certain exemplary embodiments, the SMAC/Diablo protein ishSMAC/Diablo-α having the amino acid sequence set forth in SEQ ID NO:2,encoded by the nucleic acid sequence set forth in SEQ ID NO:3.

According to certain embodiments, the SMAC/Diablo silencing moleculecomprises at least 15 consecutive nucleic acid bases substantiallyhomologous to nucleic acid bases at positions 900-1,500 of SEQ ID NO:3or to a complementary polynucleotide thereof.

According to certain embodiments, the inhibitory nucleic acid comprisesa nucleic acid sequence selected from the group consisting of SEQ IDNOs:12-17, 20-25, and 28-43, a DNA or RNA sequence correspondingthereto, analogs, derivatives and combinations thereof. Each possibilityrepresents separate embodiment of the present invention.

According to certain embodiments, the inhibitory nucleic acid isselected from the group consisting of an interfering RNA (RNAi), anantisense polynucleotide, a catalytic RNA, and an RNA-DNA chimera.

According to certain embodiments, the nucleic acid agent inhibiting theexpression of SMAC/Diablo (SMAC/Diablo silencing oligonucleotide) isselected from the group consisting of RNA interference (RNAi) moleculeand antisense molecule. According to some embodiments, the RNAi moleculeis an unmodified and/or modified double stranded (ds) RNA moleculesincluding, but not limited to, short-temporal RNA (stRNA), smallinterfering RNA (siRNA), short-hairpin RNA (shRNA), and microRNA(miRNA). In some embodiments, the SMAC/Diablo inhibitor is abiosynthetic precursor of a SMAC/Diablo-targeted dsRNA.

According to certain exemplary embodiments, the SMAC/Diablo silencingoligonucleotide is siRNA. siRNAs are typically short double-stranded RNAspecies with phosphorylated 5′ ends and hydroxylated 3′ ends. The siRNAmolecules of the invention comprise a first oligonucleotide and a secondoligonucleotide essentially complementary thereto (sense and antisenseRNA strands) that can form an RNA duplex. Typically, each strand of thesiRNA molecule is no more than 30 nucleotides in length, and ispreferably about 19-25 nucleotides in length.

The siRNA molecules may further comprise 3′ nucleotide overhangs oneither or both strands, i.e. terminal portions of the nucleotidesequence that are not base paired between the two strands of the doublestranded siRNA molecule. Preferably, the overhang is about 1-5nucleotides in length, more preferably 2 nucleotides in length.

According to certain embodiments, the SMAC/Diablo silencingoligonucleotide is siRNA molecule comprising a first oligonucleotidecomprising a nucleic acid sequence substantially homologous to at least15 consecutive nucleic acid bases of SEQ ID NO:3 and a secondpolynucleotide sequence substantially complementary to the firstoligonucleotide; wherein said first and second oligonucleotides are ableto anneal to each other to form the siRNA molecule.

According to certain exemplary embodiments, the SMAC/Diablo silencingoligonucleotide is an siRNA molecule comprising a first oligonucleotidehaving the nucleic acid sequence set forth in SEQ ID NO:12(AAGCGGUGUUUCUCAGAAUUG) and a second oligonucleotide substantiallycomplementary thereto. According to some embodiments, the secondoligonucleotide comprises the nucleic acid sequence set forth in SEQ IDNO:13 (AACAAUUCUGAGAAACCCGC). According to certain further exemplaryembodiments, the targeted SMAC/Diablo polynucleotide comprises thenucleic acid sequence set forth in any one of SEQ ID NO:3 and SEQ IDNO:5.

According to certain exemplary embodiments, the SMAC/Diablo silencingoligonucleotide is an siRNA molecule comprising a first oligonucleotidehaving the nucleic acid sequence set forth in SEQ ID NO:14(GCAGAUCAGGCCUCUAUAA) and a second oligonucleotide substantiallycomplementary thereto. According to some embodiments, the secondoligonucleotide comprises the nucleic acid sequence set forth in SEQ IDNO:15 (UUAUAGAGGCCUGAUCUGC). According to certain further exemplaryembodiments, the targeted SMAC/Diablo polynucleotide comprises thenucleic acid sequence set forth in any one of SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11.

According to certain exemplary embodiments, the SMAC/Diablo silencingoligonucleotide is an siRNA molecule comprising a first oligonucleotidehaving the nucleic acid sequence set forth in SEQ ID NO:16(CCCGGAAAGCAGAAACCAA) and a second oligonucleotide substantiallycomplementary thereto. According to some embodiments, the secondoligonucleotide comprises the nucleic acid sequence set forth in SEQ IDNO:17 (UUGGUUUCUGCUUUCCGGG). According to certain further exemplaryembodiments, the targeted SMAC/Diablo polynucleotide comprises thenucleic acid sequence set forth in any one of SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11.

According to certain embodiments, the first and/or the secondoligonucleotide of the siRNA molecules of the present invention furthercomprises 3′ overhang of two thymine nucleotides. According to theseembodiments, the SMAC/Diablo siRNA molecule is selected from the groupconsisting of an siRNA molecule comprising a first nucleotide having thenucleic acid sequence set forth in SEQ ID NO:20 and a secondoligonucleotide having the nucleic acid sequence set forth in SEQ IDNO:21; an siRNA molecule comprising a first nucleotide having thenucleic acid sequence set forth in SEQ ID NO:22 and a secondoligonucleotide having the nucleic acid sequence set forth in SEQ IDNO:23; and an siRNA molecule comprising a first nucleotide having thenucleic acid sequence set forth in SEQ ID NO:24 and a secondoligonucleotide having the nucleic acid sequence set forth in SEQ IDNO:25. Each possibility represents a separate embodiment of the presentinvention.

According to certain embodiments, the SMAC/Diablo silencingoligonucleotide is chemically modified. According to some embodiments,the SMAC/Diablo silencing oligonucleotide comprises at least one2′-sugar modification. In a particular embodiment, the 2′-sugarmodification is a 2′-O-methyl (2′-O-Me) modification.

According to certain embodiments, the SMAC/Diablo silencingoligonucleotide is modified (typically by a 2′-O-Me) at least at oneposition selected from the group consisting of position 3, 5, 6, 7, 9,10, 11, 16 and 21 of SEQ ID NO:12 or SEQ ID NO:20.

According to certain embodiments, the hSMAC/Diablo silencingoligonucleotide is modified (typically by a 2′-O-Me) at least at oneposition selected from the group consisting of position 6, 7, 10, 12,and 19 of SEQ ID NO:13 or SEQ ID NO:21.

According to some exemplary embodiments, the SMAC/Diablo silencingoligonucleotide is an siRNA molecule comprising a first oligonucleotidehaving the nucleic acid sequence set forth in SEQ ID NO:20 derivatizedby 2-O′-Me at positions 5, 10, 16 and 21 (SEQ ID NO:28) and a secondoligonucleotide having the nucleic acid sequence set forth in SEQ IDNO:21 derivatized by 2-O′-Me at positions at positions 6, 12 and 19 (SEQID NO:29).

According to some exemplary embodiments, the SMAC/Diablo silencingoligonucleotide is an siRNA molecule comprising a first oligonucleotidehaving the nucleic acid sequence set forth in SEQ ID NO:20 derivatizedby 2-O′-Me at positions 3, 6, 11, and 21 (SEQ ID NO:34) and a secondoligonucleotide having the nucleic acid sequence set forth in SEQ IDNO:21 derivatized by 2-O′-Me at positions at positions 6, and 19 (SEQ IDNO:35).

According to certain embodiments, the SMAC/Diablo silencingoligonucleotide is modified (typically by a 2′-O-Me) at least at oneposition selected from the group consisting of position 4, 6, 9, 10, 13,15 and 17 of SEQ ID NO:14 or SEQ ID NO:22.

According to certain embodiments, the hSMAC/Diablo silencingoligonucleotide is modified (typically by a 2′-O-Me) at least at oneposition selected from the group consisting of position 2, 4, 6, 8, 9,12, 13, 15, 17 and 18 of SEQ ID NO:15 or SEQ ID NO:23.

According to some exemplary embodiments, the SMAC/Diablo silencingoligonucleotide is an siRNA molecule comprising a first oligonucleotidehaving the nucleic acid sequence set forth in SEQ ID NO:22 derivatizedby 2-O′-Me at positions 4, 9, 13, and 17 (SEQ ID NO:38) and a secondoligonucleotide having the nucleic acid sequence set forth in SEQ IDNO:7 derivatized by 2-O′-Me at positions at positions 6, 12, and 18 (SEQID NO:39).

According to some exemplary embodiments, the SMAC/Diablo silencingoligonucleotide is an siRNA molecule comprising a first oligonucleotidehaving the nucleic acid sequence set forth in SEQ ID NO:22 derivatizedby 2-O′-Me at positions 6, 10, and 15 (SEQ ID NO:40) and a secondoligonucleotide having the nucleic acid sequence set forth in SEQ IDNO:23 derivatized by 2-O′-Me at positions at positions 2, 9, and 17 (SEQID NO:41).

According to some embodiments, the siRNA is a single-stranded shorthairpin RNA (shRNA) wherein the first oligonucleotide sequence isseparated from the second oligonucleotide sequence by a linker whichforms a loop structure upon annealing of the first and secondoligonucleotide sequences. In some embodiments the linker length isabout 3 to about 60 nucleotides.

According to certain embodiments, the method of the present inventioncomprises administering to the subject a nucleic acid construct capableof expressing at least one inhibitory nucleic acid.

According to some embodiments, the expressed inhibitory nucleic acidcomprises a sequence selected from the group consisting of SEQ IDNOs:12-17, 20-25, 28-29, 34-35 and 38-41. According to certainembodiments, the nucleic acid construct is capable of expressing atleast one siRNA molecule. According to certain exemplary embodiments,the method comprises administrating to the subject a construct capableof expressing siRNA molecule comprising a first oligonucleotide havingthe nucleic acid sequence set forth in SEQ ID NO:12 or SEQ ID NO:20 anda second oligonucleotide having the nucleic acid sequence set forth inSEQ ID NO:13 or SEQ ID NO:21.

The present invention discloses novel peptides derived fromhSMAC/Diablo-associated proteins that can bind to hSMAC/Diablo. When thepeptide derived from hSMAC/Diablo-interacting protein is targeted intothe cell and further into the nucleus and/or the mitochondria, such thatthe interaction with hSMAC/Diablo occurs within the organelle(s), theinteraction results in inhibition or intervention in phospholipidbiosynthesis, cell growth and/or cell proliferation.

According to further embodiments of the invention, the agent thatinhibits the activity of SMAC/Diablo is an inhibitory peptide targetedto the nucleus and/or to the mitochondria of a cell. According tocertain embodiments, the cell is overexpressing SMAC/Diablo.

According to certain embodiments, the inhibiting peptide is a conjugatecomprising a peptide derived from hSMAC/Diablo-interacting proteinhaving an amino acid sequence at least 80% homologous to an amino acidsequence selected from the group consisting of SEQ ID NOs:46-67,analogs, derivatives and/or fragments thereof, and a mitochondria and/ornucleus targeting moiety. The mitochondria and/or nucleus targetingmoiety can be peptidic or non-peptidic, and is covalently linked to thepeptide derived from SMAC/Diablo-interacting protein directly or vialinker. The targeting moiety may be linked to the peptide derived fromhSMAC/Diablo-interacting protein at any position. According to someembodiments, the targeting moiety is linked to the N- or the C-terminusof the peptide derived from hSMAC/Diablo-interacting protein. When theconjugate comprises a combination of mitochondria and nucleus targetingmoieties each moiety may be independently linked to the N- or C-terminusof the peptide or the mitochondria and nucleus targeting moieties can belinked in tandem to the N- or C-terminus of the peptide. According tosome embodiments, the conjugate further comprises a cell penetrationmoiety enhancing the permeability of the conjugate through the cellplasma membrane. Any cell penetrating moiety as is known in the art canbe used according to the teachings of the present invention.

According to some embodiments, the inhibiting peptide is a conjugate ofa peptide derived from hSMAC/Diablo-interacting protein having an aminoacid sequence at least 80% homologous to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:46-67, analogs, derivativesand/or fragments thereof, and a nucleus targeting moiety. Any nucleustargeting (localization) moiety as is known in the art can be usedaccording to the teachings of the present invention. According tocertain exemplary embodiments, the nucleus targeting moiety is thetetrapeptide RrRK, wherein r is D-arginine (SEQ ID NO:68). The nucleustargeting tetrapeptide can be independently located at the C- orN-terminus of the peptide derived from hSMAC/Diablo-interacting protein.

According to some embodiments, the inhibiting peptide is a conjugate ofa peptide derived from hSMAC/Diablo-interacting protein having an aminoacid sequence at least 80% homologous to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:46-67, analogs, derivativesand/or fragments thereof, and a mitochondria targeting moiety. Anymitochondria targeting (localization) moiety as is known in the art canbe used according to the teachings of the present invention. Accordingto certain exemplary embodiments, the mitochondria targeting moietycomprises the amino acid sequence set forth in SEQ ID NO:72. Themitochondria targeting peptide can be independently located at the C- orN-terminus of the peptide derived from hSMAC/Diablo-interacting protein.When the mitochondria targeting peptide having SEQ ID NO:72 is locatedat the C-terminus of the peptide derived from hSMAC/Diablo-interactingprotein, its C-terminus may be amidated.

According to certain embodiments, the peptide derived fromhSMAC/Diablo-interacting protein comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs:46-67. According tosome embodiments, the peptide derived from hSMAC/Diablo-interactingprotein comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49.Each possibility represents a separate embodiment of the presentinvention.

According to certain exemplary embodiments, the inhibiting peptideconjugate targeted to the nucleus comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:69, SEQ ID NO: 70 andSEQ ID NO:71.

According to certain exemplary embodiments, the inhibiting peptideconjugate targeted to the mitochondria comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:73 and SEQ ID NO:74.

According to certain embodiments, the cancer is selected from the groupconsisting of lung cancer, breast cancer, colon cancer, lymphoma,sarcomas, stomach cancer, skin cancer, renal cancer, prostate cancer,testicular cancer, cervical cancer, leukemia and pancreatic cancer. Eachpossibility represents a separate embodiment of the present invention.

According to certain currently exemplary embodiments, the cancer is lungcancer.

According to certain additional currently exemplary embodiments, thecancer is breast cancer.

According to certain embodiments treating the cancer comprisesre-programming the cancerous cells to at least one of attenuated lipidsynthesis, decreased proliferation, and differentiation.

According to certain exemplary embodiments, the cancer is lung cancerand treating the lung cancer comprises at least one of inducingdifferentiation of AT2-like cells to differentiated non-proliferatingAT1 cells; reducing proliferation of AT2-like undifferentiated cells anda combination thereof. These activities lead to the appearance ofalveolar healthy-like morphology.

Any method as is known in the art for administering the agent thatinhibits the expression and/or activity of the SMAC/Diablo can be usedaccording to the teachings of the present invention. According to someembodiment, the inhibitory agent is administered within a pharmaceuticalcomposition. According to certain exemplary embodiments, thepharmaceutical composition further comprises pharmaceutically acceptableexcipients, diluents or carriers.

According to certain embodiments, the inhibitory agent is an RNAinhibitory molecule or a peptide. According to certain currentlyexemplary embodiments, the inhibitory agent is siRNA molecule. Accordingto additional certain currently exemplary embodiments, the inhibitoryagent is a peptide conjugate comprising a peptide derived fromhSMAC/Diablo-interacting protein and a nucleus and/or mitochondriatargeting moiety. According to these embodiments, the RNAi molecule, theconstruct comprising same or the peptide conjugate is administeredwithin a pharmaceutical composition, said pharmaceutical compositionoptionally further comprises pharmaceutically acceptable excipients,diluents or carriers.

According to additional exemplary embodiments, the RNAi molecule isencapsulated within a nanoparticle or a liposome. According to someembodiments, the RNAi molecule is encapsulated within Polyethylenimine(PEI)-Poly(D,L-lactide-co-glycolide) (PLGA) nanoparticle or any othersiRNA-delivery system as is known in the art.

The mode of administering the inhibitory agent according to theteachings of the present invention will depend upon the type of theagent, the type and severity of the cancer and parameters related to thesubject (age, gender, weight etc.).

According to certain embodiments, the inhibitory agent or apharmaceutical composition comprising same is administered viaintravenous, intradermal, intramuscular, intra-arterial, intralesional,percutaneous, subcutaneous, intranasal or oral administration or byinhalation or by aerosol administration, or by combinations thereof. Insome embodiments, administration is prophylactic administration, and inalternative embodiments, administration is therapeutic administration.Each possibility represents a separate embodiment of the presentinvention.

According to certain embodiments, the method of the present inventionfurther comprises administering to the subject at least one additionalanti-cancer agent or anti-cancer therapy.

Any agent and/or therapy as is known in the art for treating thecancerous disease of the subject can be employed as long as its activitydoes not interfere with the inhibition of SMAC/Diablo expression and/oractivity according to the teachings of the present invention.

According to another aspect, the present invention provides at least oneagent that inhibits the expression and/or activity of SMAC/Diablo foruse in treating cancer associated with over-expression of SMAC/Diablo.

According to additional aspect, the present invention provides the useof at least one agent that inhibits the expression and/or activity ofSMAC/Diablo for the preparation of a medicament for treating cancerassociated with over-expression of SMAC/Diablo.

The at least one agent and the cancer types are as describedhereinabove.

According to further aspect, the present invention provides an isolatedSMAC/Diablo silencing molecule comprising a first oligonucleotidecomprising the nucleic acid sequence set forth in SEQ ID NO:12 andoptionally a 3′ overhang of 1-5 nucleotides, derivatized by 2′-O-methyl(2′-O-Me) at positions 5, 10, 16, and 21 and a second oligonucleotidecomprising the nucleic acid sequence set forth in SEQ ID NO:13 andoptionally a 3′ overhang of 1-5 nucleotides, derivatized by 2′-O-Me atpositions 6, 12 and 19.

According to certain embodiments, the SMAC/Diablo silencingoligonucleotide comprising a first oligonucleotide comprising thenucleic acid sequence set forth in SEQ ID NO:20 derivatized by 2′-O-Meat positions 5, 10, 16, and 21 and a second oligonucleotide comprisingthe nucleic acid sequence set forth in SEQ ID NO:21 derivatized by2′-O-Me at positions 6, 12 and 19.

According to yet further aspect, the present invention provides anisolated SMAC/Diablo silencing molecule comprising a firstoligonucleotide comprising the nucleic acid sequence set forth in SEQ IDNO:12 and optionally a 3′ overhang of 1-5 nucleotides, derivatized by2′-O-methyl (2′-O-Me) at positions 3, 6, 11 and 21, and a secondoligonucleotide comprising the nucleic acid sequence set forth in SEQ IDNO:13 and optionally a 3′ overhang of 1-5 nucleotides, derivatized by2′-O-Me at positions 6 and 19.

According to certain embodiments, the SMAC/Diablo silencing moleculecomprises a first oligonucleotide comprising the nucleic acid sequenceset forth in SEQ ID NO:20 derivatized by 2′-O-Me at positions 3, 6, 11,and 21 and a second oligonucleotide comprising the nucleic acid sequenceset forth in SEQ ID NO:21 derivatized by 2′-O-Me at positions 6 and 19.

According to additional aspect, the present invention provides anisolated SMAC/Diablo silencing molecule comprises a firstoligonucleotide having the nucleic acid sequence set forth in SEQ IDNO:14 and optionally a 3′ overhang of 1-5 nucleotides and a secondoligonucleotide having the nucleic acid sequence set forth in SEQ IDNO:15 and optionally a 3′ overhang of 1-5 nucleotides. According tocertain embodiments, the first oligonucleotide is derivatized by 2′-O-Meat positions 4, 9, 13 and 17 and the second oligonucleotide isderivatized by 2′-O-Me at positions 6, 12 and 18. According to otherembodiments, the first oligonucleotide is derivatized by 2′-O-Me atpositions 6, 10, and 15 and the second oligonucleotide is derivatized by2′-O-Me at positions 2, 9 and 17.

According to some embodiments, the isolated SMAC/Diablo silencingmolecule comprises a first oligonucleotide having the nucleic acidsequence set forth in SEQ ID NO:22 and a second oligonucleotide havingthe nucleic acid sequence set forth in SEQ ID NO:23. According tocertain embodiments, the first oligonucleotide is derivatized by 2′-O-Meat positions 4, 9, 13 and 17 and the second oligonucleotide isderivatized by 2′-O-Me at positions 6, 12 and 18. According to otherembodiments, the first oligonucleotide is derivatized by 2′-O-Me atpositions 6, 10, and 15 and the second oligonucleotide is derivatized by2′-O-Me at positions 2, 9 and 17.

According to additional aspect, the present invention provides anisolated SMAC/Diablo silencing molecule comprises a firstoligonucleotide having the nucleic acid sequence set forth in SEQ IDNO:16 and optionally a 3′ overhang of 1-5 nucleotides and a secondoligonucleotide having the nucleic acid sequence set forth in SEQ IDNO:17 and optionally a 3′ overhang of 1-5 nucleotides. According tocertain embodiments, the first oligonucleotide comprises the nucleicacid sequence set forth in SEQ ID NO:24 and the second oligonucleotidecomprises the nucleic acid sequence set forth in SEQ ID NO:25.

According to yet additional aspect, the present invention provides anisolated synthetic or recombinant peptide having an amino acid sequenceat least 80% homologous to an amino acid sequence selected from thegroup consisting of SEQ ID NOs:46-67, an analog, derivative or afragment thereof, wherein the peptide is capable of binding to humanSMAC/Diablo. According to certain embodiments, the peptide comprises theamino acid sequence set forth in any one of SEQ ID NOs:46-67.

According to certain embodiments, the peptide further comprises anucleus and/or mitochondria targeting moiety. The nucleus ormitochondria targeting moiety can be linked to the peptide at anyposition, typically at the N- or at the C-terminus, directly or via alinker. According to some embodiments, the conjugate further comprises acell-penetrating moiety.

According to certain embodiments, the present invention provides apeptide conjugate comprising a peptide having an amino acid sequence atleast 80% homologous to an amino acid sequence selected from the groupconsisting of SEQ ID NOs:46-67, an analog, derivative or a fragmentthereof, and a nucleus targeting peptide having the amino acid sequenceset forth in SEQ ID NO:68. According to some embodiments, the peptideconjugate comprises a peptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:46-67, an analog, derivative ora fragment thereof, and a nucleus targeting peptide having the aminoacid sequence set forth IN SEQ ID NO:68. According to certain exemplaryembodiments, the peptide conjugate comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 69, SEQ ID NO:70, andSEQ ID NO:71. Each possibility represents a separate embodiment of thepresent invention.

According to certain embodiments, the present invention provides apeptide conjugate comprising a peptide having an amino acid sequence atleast 80% homologous to an amino acid sequence selected from the groupconsisting of SEQ ID NOs:46-67, an analog, derivative or a fragmentthereof, and a mitochondria targeting peptide having the amino acidsequence set forth IN SEQ ID NO:72. According to some embodiments, thepeptide conjugate comprises a peptide having an amino acid sequenceselected from the group consisting of SEQ ID NOs:46-67, an analog,derivative or a fragment thereof, and a nucleus targeting peptide havingthe amino acid sequence set forth IN SEQ ID NO:72. According to certainexemplary embodiments, the peptide conjugate comprises an amino acidsequence selected from the group consisting of SEQ ID NO:73, and SEQ IDNO:74. Each possibility represents a separate embodiment of the presentinvention.

It is to be understood explicitly that the scope of the presentinvention encompasses homologs, analogs, variants and derivatives,including shorter and longer peptides, polypeptides, proteins andpolynucleotides, as well as peptides, polypeptides, proteins andpolynucleotide analogs with one or more amino acid or nucleic acidsubstitution, as well as amino acid or nucleic acid derivatives,non-natural amino or nucleic acids and synthetic amino or nucleic acidsas are known in the art, with the stipulation that these variants andmodifications must preserve the capacity of inhibiting the expressionand/or the activity of SMAC/Diablo according to the teachings of thepresent invention.

It is to be understood that any combination of each of the aspects andthe embodiments disclosed herein is explicitly encompassed within thedisclosure of the present invention.

Other objects, features and advantages of the present invention willbecome clear from the following description and drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates SMAC/Diablo over-expression in various types oftumors and cell lines: (A) Representative IHC staining of SMAC/Diablo innormal (n=5) and cancerous (n=20) tissue samples from tissue microarrayslides (Biomax). The percentages of samples that stained at theindicated intensities are denoted; (B) Representative immunoblotstaining of tissue lysates of healthy (H) and tumor (T) tissues withanti-SMAC/Diablo antibodies, with each pair of samples (H,T) beingderived from the same patient lung; (C) Representative immunoblotsshowing SMAC/Diablo expression in PBMCs derived from CLL patients orhealthy donors. As a loading control, actin levels were probed usinganti-β-actin antibodies; (D) Quantitative analysis of SMAC/Diabloexpression levels indicating the fold increase in the NSCLC tumor, incomparison to healthy tissue from the same patient (n=20), and in PBMCsfrom CLL patients, as compared to healthy donors (n=32); and (E, F)SMAC/Diablo expression levels in different cell lines, with the levelsin the cancer cells being presented relative to those in thenon-cancerous cells (bottom of the blot).

FIG. 2 demonstrates inhibition of cell growth resulted from silencing ofSMAC/Diablo using si-hSMAC-A: (A) The indicated cancer cell lines weretransfected with si-hSMAC-A (50 nM) and 48 h post-transfection, levelsof SMAC/Diablo in the cells were evaluated by immunoblotting FIG. 2A).β-actin was used as a loading control; (B, D) A549 and H358 cells (B)and HeLa cells (D) were transfected with si-NT or si-hSMAC-A and at theindicated time, cells were harvested and analyzed for SMAC/Diablo levelsby immunoblotting. β-actin was used as a loading control. (C, E)Quantitative analysis of the immunoblots of A549 and H358 cells (C) andHeLa cells (E) represented SMAC/Diablo level relative to the respectiveuntreated cells , at 24 h (black bar), 48 h (dark gray bar), 72 h (lightgray bar) and 96 h (white bar) (n=3); (F) HeLa, A549 and H358 cells wereuntreated (●), transfected with non-targeted siRNA (si-NT) (∘) orSMAC/Diablo-A targeted siRNA (si-hSMAC-A) (50 nM) (▴). Cell growth wasassayed at the indicated times using the SRB method (n=3); (G, H) TheA549, H358, WI-38 and HaCaT cell lines were transfected with theindicated concentration (10-50 nM) of si-NT or si-hSMAC-A. After 48 h,(G) SMAC/Diablo levels in the cells were analyzed by immunoblotting and(H) cell growth was assayed; (I) A549 cells were transfected with si-NTor si-hSMAC B, C, or D (50 nM) and at the indicated times, cells wereharvested and analyzed for SMAC/Diablo levels by immunoblotting. β-actinwas used as a loading control. (J) Bar graph represents inhibition ofgrowth of A549 cells treated with si-NT or si-hSMAC B, C, or D (50 nM).(K) Quantitative analysis of Ki-67 positive cells of si-NT- orsi-hSMAC-A (50 nM)-treated A549 cells, as analyzed fromimmunofluorescence staining for Ki-67 (L) Cell cycle analysis of A549cells treated with si-hSMAC (50 nM) by flow cytometry. Cells incubatedwith etoposide (10 μM, 24 h) were used as positive control. (M) ATPlevels were analyzed at 24, 48 and 72 h post-transfection in HeLa (blackbar), A549 (dark gray bar) and H358 cells (light gray bar) treated withsi-NT or si-hSMAC-A (50 nM).

FIG. 3 depicts cell death induced by overexpression of exogenousSMAC/Diablo: (A) Bar-graph represents % of cell death induced by si-NTor si-hSMAC-A (50 nM) or selenite (5 μM) in HeLa, A549 and H358 cells.Cells were transfected with si-NT or si-hSMAC-A and analyzed for celldeath after 24 h (white bar), 48 h (black bar) and 72 h (gray bar) usingpropidium iodide (PI) staining by flow cytometry. Selenite (5 μM) usedas positive control; (B) Representative cell death analysis of A549cells treated with si-NT or si-hSMAC-A (50 nM, 48 h) or selenite (10 μM,24 h) using FITC-Annexin V/PI staining and flow cytometry. (C)Qantitative analysis of cell deathin cells treated for the indicatedtime with 50 nM si-NT (black bar) or si-hSMAC-A (gray bar) or seleniteusing FITC-Annexin V/PI staining by flow cytometry. (D, E)Representative immunoblot staining by anti-SMAC/Diablo antibodies ofcell lysate from HeLa, A549 and H358 cells 48 h of post-transfectionwith pEGFP encoding plasmid (D) or SMAC/Diablo-GFPpcDNA 3.1 plasmid(0.75 μg) (E); (F) Bar-graph representing % of cell death induced by GFPor SMAC/Diablo-GFPpcDNA 3.1 plasmid (1 μg-2 μg) in HeLa, A549 and H358cells. Cells death was analyzed after 24 and 48 hour using PI stainingby flow cytometry.

FIG. 4 depicts inhibition by si-hSMAC-A of tumor growth of lung cancerxenografts: (A) A549 cells were inoculated into nude mice (3×10⁶cells/mouse). Tumor volumes were monitored, and on day 18, mice withsimilar average tumor volumes (75-90 mm³) were divided into three groups(n=5). Xenografts were injected with si-NT (●, 50 nM) or si-hSMAC (350nM (∘) or 700 nM (▴)). The size of the xenografts was measured andaverage tumor volumes are presented as means±SEM, p:**≤0.01; p:***≤0.001; (B) Representative tumors; (C) Weights of dissected tumorsfrom mouse A549 cell xenografts after treatment with si-NT (si-NTtreated tumors, si-NT-TTs) or si-hSMAC-A (si-hSMAC treated tumors,si-hSMAC-A-TTs); (D) Representative sections from si-NT-TTs andsi-hSMAC-A-TTs stained with anti-SMAC/Diablo antibodies; (E) Expressionof α- and ε-SMAC/Diablo isoforms in RNA isolated from si-NT- andsi-hSMAC-A-TTs using PCR and specific primers; (F) Representativesections from si-NT-TTs and si-hSMAC-A-TTs stained with anti-Ki-67antibodies; and (G) Quantitative analysis of Ki-67 protein (grey bars)and mRNA (q-PCR) (black bars) levels in si-NT- and si-hSMAC-A-TTs.

FIG. 5 depicts that si-hSMAC-A inhibited cell growth in vitro and tumorgrowth of breast cancer xenografts: (A) MDA-MB-231 cells weretransfected with si-NT (50 nM) or si-hSMAC-A (100 nM) and analyzed forcell growth at the indicated time using the SRB method (n=3) (Control(●), si-NT (∘), si-hSMAC-A (▴); (B) MDA-MB-231 cells were transfectedwith si-NT or si-hSMAC-A and at the indicated time cells were harvestedand analyzed for SMAC/Diablo levels by immunoblotting. (3-actin was usedas a loading control; (C) MDA-MB-231 cells (3×10⁶cells/mouse) wereinoculated into nude mice. Tumor volumes were monitored (using a digitalcaliper) and on day 18, the mice were divided into two groups (n=8 each)with each mouse in the group containing a tumor with a volume between 60and 100 mm³ and similar average volumes measured per group. The two micegroups were subjected to the following treatments: Xenografts wereinjected with si-NT (●, 350 nM) or si-hSMAC-A (▴, 700 nM). (D)Represents photomicrograph of dissected tumors from mouse MDA-MB-231cell xenografts treated with si-NT or si-hSMAC-A; (E) Bar-graphrepresents dissected tumors weight from MDA-MB-231 cell xenografts;calculated average tumor volumes are presented as means±SEM, ***P<0.001(F, G) Dissected tumors were subjected to immunohistochemistry.Photomicrographs represent IHC staining of tumor sections from threemice from each group with (F) anti-SMAC/Diablo and (G) anti-Ki-67antibodies. Bars represent 50 μm.

FIG. 6 depicts that silencing of SMAC/Diablo affects the expression ofits associated proteins: (A, B) mRNA levels of SMAC/Diablo, XIAP, cIAP1,cIAP2, cytochrome c, AIF caspase 8, 9 and 3 as analyzed using q-PCR insiNT-TTs relative to si-hSMAC-A-TT (presented as fold change). (C) HeLa,A549 and H358 cells were transfected with si-NT or si-hSMAC-A (50 nM)and at the indicated time cells were harvested and analyzed forSMAC/Diablo, XIAP, Caspase 8, 9 and 3 by immunoblotting. β-actin wasused as a loading control; (D) Quantitative analysis of the immunoblotwas carried out and presented (relative units (RU)) for all cell linesat the 48 h (gray bar) and 96 h (black bar) of post-transfection (n=3);(E) Bar graph represents relative mRNA levels of SMAC/Diablo, XIAP,cIAP1 and cIAP2 using q-PCR technique in HeLa, A549 and H358 cells after48 h (gray bar) and 96 h (black bar) of post-transfection withsi-hSMAC-A (50 nM).

FIG. 7 depicts the morphological changes induced in tumors treated withsi-hSMAC: (A) Representative sections from si-NT- and si-hSMAC-A-TTsstained with H&E; (B) Enlarged images of representative sections fromsi-NT- and si-hSMAC-A-TTs stained with H&E, showing glandular-likeclusters surrounded by a chain of cells (arrows) in si-hSMAC-A-TTs; (C,D, E) Sections from si-NT- and si-hSMAC-A-TTs stained withanti-prosurfactant c (C) or anti-podoplanin antibodies (D, E); (F)Representative sections from si-NT- and si-hSMAC-A-TTs stained withanti-CD31 antibodies; and (G, H) Schematic presentations of alveoli (H)and a cross-section through alveoli, with major cell types indicated(I).

FIG. 8 depicts the staining of stromal markers in si-NT-TTs andsi-hSMAC-A-TTs: (A) Representative sections from si-NT-TTs andsi-hSMAC-A-TTs stained with H&E showing stromal structures; (B) vascularformation with red blood cells (arrows) in si-NT-TTs but notsi-hSMAC-A-TTs; and (C) with anti-a-SMA antibodies.

FIG. 9 depicts the nuclear and mitochondrial localization ofSMAC/Diablo: (A) Representative sections from si-NT-TTs derived fromA549 cells stained with anti-SMAC/Diablo antibodies and viewed byimmunofluorescence. (B, C) IHC staining of SMAC/Diablo expression incancerous lung tissue from tissue microarray slides (Biomax) indicatingnuclear (B) and cytosolic (C) localization of SMAC/Diablo.

FIG. 10 depicts the differentially expressed genes and subcellularmorphological alterations induced by reductions in SMAC levels in lungcancer cell derived xenografts: NGS data analysis showing selecteddown-regulated (A) and up-regulated (B) genes associated with theextracellular matrix, including cell-secreted collagen andproteoglycans, extracellular exosomes and proteins in the endoplasmicreticulum and Golgi lumen associated with vesicle formation. The numberof genes and p-values are indicated for each category; (C) Changes (asrevealed by NGS) in the expression of genes associated with lipidtransport, synthesis, and degradation in si-hSMAC-A-TTs, represented asfold change, relative to their expression in si-NT-TTs; (D)Representative electron microscopic images of si-NT-TTs andsi-hSMAC-A-TTs sections. Arrows points to lamellar bodies; (E) Thelevels of phosphatidylcholine (PC), phosphatidylethanol amine (PE) andtotal phospholipids (PL) in si-hSMAC-A-TTs, relative to si-NT-TTs,determined as described hereinabove; and (F) Changes in the expressionof mRNA (q-PCR) associated with phosphatidylcholine synthesis insi-hSMAC-A-TTs, presented as fold change. (G) Schematic representationof diacylglycerols (a) and phosphatidylcholine synthesis (b, c)pathways, with down- and up-regulated genes identified by arrows.

FIG. 11 depicts the genes associated with transporters, metabolism,inflammatory response, exosome and vesicles formation, differentiallyexpressed between si-NT-TTs and si-hSMAC-A-TTs: results from NGS datashowing the changes in the expression of genes associated withtransporters (A) and metabolism (B) related proteins are shown as foldof changes in si-hSMAC-A-TTs relative to si-NT-TTs. (C) The changes inmRNA level (q-PCR) of the genes associated with inflammatory response,redox state regulation (RSR), and exosome are shown.

FIG. 12 depicts that EM and cell membrane network is modified insi-hSMAC-treated tumors: (A, B) Representative electron microscopicimage of si-NT and si-hSMAC-A-TTs. Arrows point to lamellar bodies inthe si-NT-TTs (A). Black and white arrows point to nuclear membrane andmitochondria, respectively (B).

FIG. 13 is a schematic representation of the effects of SMAC/Diablodepletion on tumor morphology and properties. A schematic presentationof various membrane organelles as ER, Golgi Apparatus, and the endocyticand exocytic membrane dynamics representing intra- and extra-cellulartrafficking routes in which the expression of various gens was alteredupon silencing of SMAC/Diablo expression.

FIG. 14 demonstrates growth inhibition of the of lung cancer cell lineA549 resulted from silencing of SMAC/Diablo using modified si-hSMAC-A(designated A1-A4) or modified si-hSMAC-B (designated B 1-B4) molecules.(A, D) The cells were transfected with each of the indicated si-hSMAC(30 or 50 nM) and 48 h post-transfection, levels of SMAC/Diablo in thecells were evaluated by immunoblotting. β-actin was used as a loadingcontrol. (B, E) Quantitative analysis of the immunoblots. (C, F) Growthof the cells transfected with each of the si-hSMAC at the indicatedconcentration was assayed using the SRB method.

FIG. 15 shows protein interactions of SMAC/Diablo. (A) Network analysisof human proteins interacting with SMAC (B) protein interactions ofSMAC/Diablo with glass-bound peptide array consisting of overlappingpeptides derived from 14 SMAC/Diablo-interacting proteins. The peptidearray was incubated overnight with purified SMAC/Diablo (0.8 μM) andthen blotted with anti-SMAC antibodies (1:2000), followed by incubationwith HRP-conjugated anti-mouse IgG and detection using achemiluminescence kit. The black dots indicate SMAC/Diablo binding andthe numbers their location on the array, allowing identification of thepeptide.

FIG. 16 demonstrates the effect of peptide 2C18 (SEQ ID NO:47) derivedfrom the SMAC/Diablo-interacting protein Ubiquitin conjugating enzyme E2K Protein (UBEK2) on SMAC/Diablo capability to interact with the 2C18peptide and additional peptides of the glass-bound peptide array. (A)Peptide array incubated with free SMAC/Diablo as described for FIG. 15hereinabove. (B) SMAC (0.8 μM) was incubated with the synthetic peptides2C18 (2.4 μM) and blotted as in (A). The peptide spots that eliminateddue to interaction with SMAC/Diablo are circled.

FIG. 17 demonstrates the effect of peptide 1G12 (SEQ ID NO:48) derivedfrom the SMAC/Diablo-interacting protein Baculoviral IAP repeatcontaining 2 Protein (BIRC2) on SMAC/Diablo capability to interact withthe 1G12 peptide and additional peptides of the glass-bound peptidearray. (A) Peptide array incubated with free SMAC/Diablo as describedfor FIG. 15 hereinabove. (B) SMAC (0.8 μM) was incubated with thesynthetic peptides derived from BIRC2 spot 1G12 (2.4 μM) blotted as in(A). The peptide spots that eliminated due to interaction withSMAC/Diablo are circled.

FIG. 18 shows that SMAC/Diablo interacts with phosphatidylserinedecarboxylase (PSD/PISD). (A) SMAC and PSD purification: lane 1,bacteria extract (Crude ext); lane 2 partially purified PSD (Step 1);lane, 3, purified SMAC; and lane 4, purified PSD (step 2). (B) PSDactivity measured by following the formation of phosphatidylethanolamine(PE). (C) PSD binding to SMAC/Diablo using the microscale thermophoresisMST method. (D). A model of mitochondria with proposed SMAC functions inthe regulation of PE synthesis at ER-mitochondria contact sites. PS isproduced in the ER from PC and PE, and PS then is transferred to themitochondria, where it is converted by PSD into PE.

DETAILED DESCRIPTION OF THE INVENTION

The present invention addresses the paradox of overexpression ofSMAC/Diablo, a protein participating in promoting apoptosis bynegatively regulating the inhibitor of apoptosis protein (IAP) family,in several types of cancer. The present invention shows for the firsttime that silencing SMAC/Diablo expression and/or modulating itsactivity within the nucleus and/or mitochondria in cancerous cellsover-expressing this protein, reduced the proliferation of the cellsand/or the growth of tumors comprising cells with the modulatedexpression and or activity of SMAC/Diablo. According to someembodiments, SMAC/Diablo expression is silenced by RNA inhibitorymolecules and its activity within the nucleus and/or mitochondria ismodulated using peptides derived with proteins interacting withSMAC/Diablo.

Using as example lung cancer xenografts treated with SMAC/Diablosilencing siRNA molecule, immunohistochemistry and electron-microscopyof residual lung tumor tissue demonstrated morphological changes,including the development of glandular, alveoli-like structures andelimination of lamellar bodies. Next-generation sequencing of lungtumors treated with SMAC/Diablo specific siRNA molecule revealed alteredexpression of genes associated with intercellular membranal and exosomalnetworks, cells differentiation, lipid metabolism and transporterexpression and/or activity compared to lung tumors treated withnon-targeted-siRNA molecule. Silencing of SMAC/Diablo expressiondecreased phospholipids and phosphatidylcholine levels, and theexpression of enzymes associated with their synthesis. Without wishingto be bound by any specific theory or mechanism of action, theabove-described phenomena suggest that SMAC/Diablo possesses anadditional, non-apoptotic function associated with, inter alia,regulation of lipid synthesis essential for cancer cell growth. Agentsinhibiting the expression and/or activity of SMAC/Diablo within thenucleus and/or the mitochondria in cancer types overexpressing thisprotein can thus be used as therapeutics.

Definitions

As used herein, the terms “SMAC/Diablo” and “SMAC” are used hereininterchangeably and refer to the second mitochondria-derived activatorof caspase/direct inhibitor of apoptosis-binding protein with low pIprotein. Several SMAC/Diablo variants, generated by alternativesplicing, are known in the art, all encompassed within the teachings ofthe present invention. According to certain embodiments, the terms asused herein refer to a protein having an amino acid sequence at least80% homologous to the human SMAC/Diablo (hSMAC/Diablo) protein having anamino acids sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO: 6, SEQ ID NO:8 and SEQ ID NO:10. According to certainexemplary embodiments, the terms refers to hSMAC/Diablo protein havingan amino acids sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO:8 and SEQ ID NO:10.

As used herein, the term “treatment” or “treating” refers to clinicalintervention designed to alter the natural course of the individual orcell being treated during the course of clinical pathology, e.g.,cancer. Desirable effects of treatment include decreasing the rate ofdisease progression (delaying progression of a disease), ameliorating orpalliating the disease state, and remission or improved prognosis of thedisease. For example, an individual is successfully “treated” if one ormore symptoms associated with cancer are mitigated or eliminated,including, but are not limited to, reducing the proliferation ofcancerous cells, decreasing symptoms resulting from the disease,increasing the quality of life of those suffering from the disease,decreasing the dose of other medications required to treat the disease,delaying the progression of the disease, and/or prolonging survival ofindividuals.

As used herein, “delaying progression of a disease” means to defer,hinder, slow, retard, stabilize, and/or postpone development of thedisease (such as cancer). This delay can be of varying lengths of time,depending on the history of the disease and/or individual being treated.As is evident to one skilled in the art, a sufficient or significantdelay can, in effect, encompass prevention, in that the individual doesnot develop the disease. For example, a late stage cancer, such asdevelopment of metastasis, may be delayed.

The terms “cell growth” and “cell proliferation” are used hereininterchangeably and refer to the number of viable cells of a particulartype observed after a certain growth period.

The terms “inhibit” “decrease”, “reduce” and ‘silence” with regard tothe expression or activity of SMAC/Diablo are used hereininterchangeably and includes any decrease in expression or proteinactivity or level of the SMAC/Diablo gene or mRNA or protein encoded bythe SMAC/Diablo. According to certain embodiments, inhibition ofSMAC/Diablo activity refers to modulation or inhibition of SMAC/Diabloassociation with partner proteins within the nucleus and/ormitochondria, resulting in an inhibition/decrease/reduction of cellgrowth (proliferation). The decrease may be of at least 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to theexpression of a target gene/mRNA or level of the protein encoded by agene which has not been targeted by an inhibiting agent and/or ascompared to the proliferation rate of cells in which the targetgene/mRNA was not silenced or of cells in which the activity of theprotein was not modulated.

The terms “polynucleotide”, “nucleic acid sequence”, “polynucleotidesequence” and “oligonucleotide” are used interchangeably herein andrefer to an oligomer or polymer of ribonucleic acid(ribo-oligonucleotide or ribo-oligonucleoside) or deoxyribonucleic acidcomprising up to about 100-1,000 nucleic acid residues. These termsencompass nucleotide sequences strands composed of naturally-occurringnucleobases, sugars and covalent inter-sugar linkages as well aspolynucleotides having non-naturally-occurring portions which functionsimilarly. Such modified or substituted polynucleotides may be preferredover native forms because of the valuable characteristics including, forexample, increased stability in the presence of plasma nucleases andenhanced cellular uptake. A polynucleotide may be a polymer of RNA orDNA or hybrid thereof, that is single- or double-stranded, linear orbranched, and that optionally contains synthetic, non-natural or alterednucleotide bases. The terms also encompass RNA/DNA hybrids. It is to beexplicitly understood that the polynucleotide sequences provided hereincan be of DNA or RNA molecules.

“RNA interference (RNAi)” is an evolutionally conserved process wherebythe expression or introduction of RNA of a sequence that is identical orhighly similar to a target gene results in the sequence specificdegradation or specific post-transcriptional gene silencing (PTGS) ofmessenger RNA (mRNA) transcribed from that targeted gene, therebyinhibiting expression of the target gene. In some embodiments, the RNAis double stranded RNA (dsRNA). This process has been described inplants, invertebrates, and mammalian cells. In nature, RNAi is initiatedby the dsRNA-specific endonuclease Dicer, which promotes cleavage oflong dsRNA into double-stranded fragments termed siRNAs. siRNAs areincorporated into a protein complex that recognizes and cleaves targetmRNAs. RNAi can also be initiated by introducing nucleic acid molecules,e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silencethe expression of target genes. As used herein, inhibition by RNAiincludes any decrease in expression or protein activity or level of theSMAC/Diablo gene or mRNA or protein encoded by the target gene, i.e.,SMAC/Diablo. The decrease may be of at least 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95% or 99% or more as compared to the expression of atarget gene or the activity or level of the protein encoded by a targetgene which has not been targeted by an RNA interfering agent.

“Small interfering RNA” (siRNA), also referred to herein as “shortinterfering RNA” is defined as an agent which functions to inhibitexpression of a target gene (silencing the gene) e.g., by RNAi. An siRNAmay be chemically synthesized, may be produced by in vitrotranscription, or may be produced within a host cell. In one embodiment,siRNA is a double stranded RNA (dsRNA) molecule of about 15 to about 40nucleotides in length, preferably about 15 to about 28 nucleotides, morepreferably about 19 to about 25 nucleotides in length, and morepreferably about 19, 20, 21, or 22 nucleotides in length, and maycontain a 3′ and/or 5′ overhang on each strand having a length of about0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhang isindependent between the two strands, i.e., the length of the overhang onone strand is not dependent on the length of the overhang on the secondstrand. Preferably the siRNA is capable of promoting RNA interferencethrough degradation or specific post-transcriptional gene silencing(PTGS) of the target messenger RNA (mRNA). According to otherembodiment, an siRNA is a small hairpin (also called stem loop) RNA(shRNA). In some embodiments, these shRNAs are composed of a short(e.g., 19-25 nucleotides) antisense strand, followed by a 5-9 nucleotideloop, and the analogous sense strand. Alternatively, the sense strandmay precede the nucleotide loop structure and the antisense strand mayfollow. RNA interfering agents, e.g., siRNA molecules, may beadministered to a subject having or at risk for having cancer associatedwith over-expression of SMAC/Diablo, to inhibit expression ofSMAC/Diablo, and thereby treat, ameliorate, or inhibit the cancer in thesubject.

The terms “construct”, or “RNAi expression construct” are used hereininterchangeably to describe an artificially assembled or isolatednucleic acid molecule which includes the polynucleotide of interest. Ingeneral, a construct may include the polynucleotide or polynucleotidesof interest, a marker gene which in some cases can also be a gene ofinterest and appropriate regulatory sequences. According to certainembodiments of the invention, the polynucleotide of interest encodesiRNA molecule. It should be appreciated that the inclusion ofregulatory sequences in a construct is optional, for example, suchsequences may not be required in situations where the regulatorysequences of a host cell are to be used. The regulatory elementstypically include a promoter sequence for directing transcription of thepolynucleotide of interest in the cell in a constitutive or induciblemanner. The term construct includes vectors but should not be seen asbeing limited thereto. According to certain embodiments, the term“vector,” is intended to refer to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid,” which refers to a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a phage vector. Another type of vector is a viral vector,wherein additional DNA segments may be ligated into the viral genome(such as an adenoviral vector, a lentiviral vector, etc.). Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors having a bacterial originof replication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell and thereby arereplicated along with, the host genome.

The terms “complementary” or “complement thereof” are used herein torefer to the sequence of polynucleotide which is capable of formingWatson & Crick base pairing with another specified polynucleotidethroughout the entirety of the complementary region. This term isapplied to pairs of polynucleotides based solely upon their sequencesand not any particular set of conditions under which the twopolynucleotides would actually bind. The terms “substantiallycomplementary” and “sufficiently complementary” are used hereininterchangeably. An oligomeric compound need not be 100% complementaryto its target nucleic acid to be specifically hybridizable. Moreover, anoligomeric compound may hybridize over one or more segments such thatintervening or adjacent segments are not involved in the hybridization(e.g., a bulge, a loop structure or a hairpin, structure). A“non-complementary nucleobase” means a nucleobase of an antisenseoligonucleotide that is unable to undergo precise base pairing with anucleobase at a corresponding position in a target nucleic acid. In someembodiments there are non-complementary positions, also known as“mismatches”, between the oligomeric compound and the target nucleicacid, and such non-complementary positions may be tolerated between anoligomeric compound and the target nucleic acid provided that theoligomeric compound remains substantially complementary to the targetnucleic acid.

As used herein, the term “peptide” indicates a sequence of amino acidslinked by peptide bonds. Peptides according to some embodiments of thepresent invention consist of 10-50 amino acids, for example 15-35 aminoacids or 20-25 amino acids.

In some embodiments, a peptide according to the present invention is upto 30 amino acids, for example up to 29 amino acids, 28 amino acids, 27amino acids, 26 amino acids, 25 amino acids, 24 amino acids, 23 aminoacids, 22 amino acids, 21 amino acids, 20 amino acids, 19 amino acids,18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 aminoacids, 13 amino acids, 12 amino acids, 11 amino acids, or up to 10 aminoacids. Each possibility represents a separate embodiment of theinvention.

The term “amino acid” refers to compounds, which have an amino group anda carboxylic acid group, preferably in a 1,2-1,3-, or 1,4-substitutionpattern on a carbon backbone. α-Amino acids are most preferred, andinclude the 20 natural amino acids (which are L-amino acids except forglycine) which are found in proteins, the corresponding D-amino acids,the corresponding N-methyl amino acids, side chain modified amino acids,the biosynthetically available amino acids which are not found inproteins (e.g., 4-hydroxy-proline, 5-hydroxy-lysine, citrulline,ornithine (Orn), canavanine, djenkolic acid, β-cyanoalanine), andsynthetically derived α-amino acids, such as aminoisobutyric acid,norleucine (Nle), norvaline (NorVal, Nva), homocysteine and homoserine.β-Alanine and γ-amino butyric acid are examples of 1,3 and 1,4-aminoacids, respectively, and many others as well known to the art.

Some of the amino acids used in this invention are those which areavailable commercially or are available by routine synthetic methods.Certain residues may require special methods for incorporation into thepeptide, and either sequential, divergent or convergent syntheticapproaches to the peptide sequence are useful in this invention. Naturalcoded amino acids and their derivatives are represented by one-lettercodes or three-letter codes according to IUPAC conventions. When thereis no indication, the L isomer was used. The D isomers are indicated by“D” or “(D)” before the residue abbreviation.

As used herein, an “amino acid residue” means the moiety which remainsafter the amino acid has been conjugated to additional amino acid(s) toform a peptide, or to a moiety (such as a cell penetrating moiety (CPP)and/or mitochondria and/or nucleus targeting moiety), typically throughthe alpha-amino and carboxyl of the amino acid.

As used herein, the terms “targeting moiety” and “localization moiety”with reference to targeting of a peptide of the invention to the nucleusand/or mitochondria are used herein interchangeably and refer to amolecule which is able to target the peptide to the specific organelleand facilitate or enhance its penetration into the nucleus ormitochondria. The targeting moiety typically enhances the permeabilityof the peptide, i.e. its ability to penetrate, pervade, or diffusethrough a barrier or membrane, typically a phospholipid membrane. Thenucleus and/or mitochondria targeting moiety may also enhance thepenetration of the peptide through the plasma membrane. Additionally oralternatively, a cell penetrating moiety (CPP) specifically designed toenhance the permeability of the peptide through the plasma membrane isadded to the targeting moiety.

The term “expression”, as used herein, refers to the production of afunctional end-product e.g., an mRNA or a protein.

According to one aspect, the present invention provides a method fortreating or delaying progression of cancer associated withover-expression of second mitochondria-derived activator ofcaspase/direct inhibitor of apoptosis-binding protein with low pI(SMAC/Diablo) in a subject in need thereof, comprising administering tothe subject an effective amount of at least one agent that decreases orinhibits the expression of SMAC/Diablo gene and/or protein and/ormodulate the protein activity, and/or its interaction with associatedproteins (partners).

According to certain embodiments, the agent that decreases or inhibitsthe expression and/or activity of SMAC/Diablo is selected from the groupconsisting of an inhibitory nucleic acid, an inhibitory small molecule,an inhibitory polypeptide or peptide and an inhibitory aptamer.

According to certain aspects, the present invention provides a methodfor treating or delaying progression of cancer associated withover-expression of second mitochondria-derived activator ofcaspase/direct inhibitor of apoptosis-binding protein with low pI(SMAC/Diablo) in a subject in need thereof, comprising administering tothe subject an effective amount of at least one RNAi molecule, the RNAimolecule comprises a nucleic acid sequence set forth in any one of SEQID NOs:12-43.

According to certain embodiments, the method comprises administering aneffective amount of an RNAi molecule comprises a nucleic acid sequenceset forth in any one of SEQ ID NOs:12-17, 20-25, 28-29, 34-35, and38-42. Each possibility represents a separate embodiment of the presentinvention.

Inhibitory Nucleic Acids

According to certain embodiments, the agent is an inhibitory nucleicacid Nucleic acid inhibitors can be used to decrease the expression ofSMAC/Diablo gene. Nucleic acid inhibitors include a siRNA, a dsRNA, aribozyme, a triple-helix former, an aptamer, or an antisense nucleicacid. siRNAs are small double stranded RNAs (dsRNAs) as describedhereinabove. Antisense agents can include, for example, from about 8 toabout 80 nucleobases (i.e. from about 8 to about 80 nucleotides), e.g.,about 8 to about 50 nucleobases, or about 12 to about 30 nucleobases.Antisense compounds include ribozymes, external guide sequence (EGS)oligonucleotides, and other short catalytic RNAs or catalyticoligonucleotides which hybridize to the target nucleic acid and modulateits expression. Antisense compounds can include a stretch of at leasteight consecutive nucleobases that are complementary to a sequence inthe target gene.

According to certain embodiments, the inhibitory nucleic acid comprisesat least 15 consecutive nucleic acids substantially identical to a geneor mRNA at least 80% homologous to the gene or mRNA encoding humanSMAC/Diablo (hSMAC/Diablo) protein or to a complementary polynucleotidethereof.

According to certain embodiments, the inhibitory nucleic acid istargeted to a SMAC/Diablo encoding gene comprising a nucleic acidsequence at least 80%, at least 85%, at least 90%, at least 95% or morehomologous to the nucleic acid sequence set forth in SEQ ID NO:1(NG_029459.1).

According to certain embodiments, the hSMAC/Diablo protein is selectedfrom the group consisting of SMAC/Diablo-α, having the amino acidsequence set forth in SEQ ID NO:2; SMAC/Diablo-δ, having the amino acidsequence set forth in SEQ ID NO:4; SMAC/Diablo-β, having the amino acidsequence set forth in SEQ ID NO:6; SMAC/Diablo-4, having the amino acidsequence set forth in SEQ ID NO:8; and SMAC/Diablo-γ, having the aminoacid sequence set forth in SEQ ID NO:10. Each possibility represents aseparate embodiment of the present invention.

According to certain embodiments, the hSMAC/Diablo-α is encoded by thenucleic acid sequence having SEQ ID NO:3; hSMAC/Diablo-δ is encoded bythe nucleic acid sequence having SEQ ID NO:5; hSMAC/Diablo-β is encodedby the nucleic acid sequence having SEQ ID NO:7; hSMAC/Diablo-4 isencoded by the nucleic acid sequence having SEQ ID NO:9; andhSMAC/Diablo-γ is encoded by the nucleic acid sequence having SEQ IDNO:11. Each possibility represents a separate embodiment of the presentinvention.

The nucleic acid agents of the present invention are of at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17 at least 18, at least 19, at least 20, or at least 21bases specifically hybridizable with SMAC/Diablo RNA, but excluding thefull length SMAC/Diablo transcript or known variants thereof. TheSMAC/Diablo-silencing oligonucleotides of the invention are preferablyno more than about 1000 bases in length, more preferably no more thanabout 100 bases in length. In other preferable embodiments, theoligonucleotides are no more than 30 nucleotides (or base pairs) inlength.

According to certain embodiments, the RNAi molecule comprises a nucleicacid sequence selected from the group consisting of SEQ ID NO:12-43.

According to certain exemplary embodiments, the SMAC/Diablo inhibitorynucleic acid is siRNA molecule selected from the group consisting of:

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:12 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:13;

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:14 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:15;

siRNA comprising a first sense strand having the nucleic acid sequenceset forth in SEQ ID NO:16 and an antisense strand having the nucleicacid sequence set forth in SEQ ID NO:17; and

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:18 and second polynucleotide having the nucleic acidsequence set forth in SEQ ID NO:19.

According to certain embodiments, the hSMAC/Diablo protein ishSMAC/Diablo-β having the amino acid sequence set forth in SEQ ID NO:6,encoded by the nucleic acid sequence set forth in NO:7. According tothese embodiments, wherein the inhibitory nucleic acid is siRNAmolecule, the siRNA molecule is selected from the group consisting ofsiRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:22 and second polynucleotide having the nucleic acidsequence set forth in SEQ ID NO:23; siRNA comprising a sense strandhaving the nucleic acid sequence set forth in SEQ ID NO:24 and anantisense strand having the nucleic acid sequence set forth in SEQ IDNO:25; and siRNA comprising a sense strand having the nucleic acidsequence set forth in SEQ ID NO:26 and an anti sense strand having thenucleic acid sequence set forth in SEQ ID NO:27.

According to certain embodiments, the hSMAC/Diablo protein ishSMAC/Diablo-δ having the amino acid sequence set forth in SEQ ID NO:4,encoded by the nucleic acid sequence set forth in SEQ ID NO:5. Accordingto these embodiments, wherein the inhibitory nucleic acid is siRNAmolecule, the siRNA molecule is selected from the group consisting ofsiRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:20 and an anti sense strand having the nucleic acidsequence set forth in SEQ ID NO:21; siRNA comprising a sense strandhaving the nucleic acid sequence set forth in SEQ ID NO:22 and anantisense strand having the nucleic acid sequence set forth in SEQ IDNO:23; and siRNA comprising a sense strand having the nucleic acidsequence set forth in SEQ ID NO:24 and an antisense strand having thenucleic acid sequence set forth in SEQ ID NO:25.

According to certain embodiments, the hSMAC/Diablo protein ishSMAC/Diablo-4 having the amino acid sequence set forth in SEQ ID NO:8,encoded by the nucleic acid sequence set forth in SEQ ID NO:9. Accordingto these embodiments, wherein the inhibitory nucleic acid is siRNAmolecule, the siRNA molecule is selected from the group consisting ofsiRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:22 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:23; siRNA comprising a sense strandhaving the nucleic acid sequence set forth in SEQ ID NO:24 and anantisense strand having the nucleic acid sequence set forth in SEQ IDNO:25; and siRNA comprising a sense strand having the nucleic acidsequence set forth in SEQ ID NO:26 and an antisense strand having thenucleic acid sequence set forth in SEQ ID NO:27.

According to certain embodiments, the hSMAC/Diablo protein ishSMAC/Diablo-γ having the amino acid sequence set forth in SEQ ID NO:10,encoded by the nucleic acid sequence set forth in SEQ ID NO:11.According to these embodiments, wherein the inhibitory nucleic acid issiRNA molecule, the siRNA molecule is selected from the group consistingof siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:22 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:23; and siRNA comprising a sense strandhaving the nucleic acid sequence set forth in SEQ ID NO:24 and anantisense strand having the nucleic acid sequence set forth in SEQ IDNO:25.

In some embodiments, the sense and antisense strands of the presentsiRNA can comprise two complementary, single-stranded RNA molecules orcan comprise a single molecule in which two complementary portions arebase-paired and are covalently linked by a single-stranded “hairpin”area. Without wishing to be bound by any theory, it is believed that thehairpin area of the latter type of siRNA molecule is cleavedintracellularly by the “Dicer” protein (or its equivalent) to form asiRNA of two individual base-paired RNA molecules.

Preferably, one or both strands of the siRNA of the invention can alsocomprise a 3′ overhang. As used herein, a “3′ overhang” refers to atleast one unpaired nucleotide extending from the 3′-end of an RNAstrand. Thus in one embodiment, the siRNA of the invention comprises atleast one 3′ overhang of from 1 to about 6 nucleotides (which includesribonucleotides or deoxynucleotides) in length, from 1 to about 5nucleotides in length, from 1 to about 4 nucleotides in length, or fromabout 2 to about 4 nucleotides in length. In the embodiment in whichboth strands of the siRNA molecule comprise a 3′ overhang, the length ofthe overhangs can be the same or different for each strand. In a mostpreferred embodiment, the 3′ overhang is present on both strands of thesiRNA, and is 2 nucleotides in length. For example, each strand of thesiRNA of the invention can comprise 3′ overhangs of dithymidylic acid(“TT”) or diuridylic acid (“UU”).

As illustrated in the Example section hereinbelow, exemplary siRNAoligonucleotides of the present invention are 19 to 21 base pairs inlength with two 3′ overhang on each strand, selected from the groupconsisting of:

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:20 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:21;

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:22 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:23;

siRNA comprising a first sense strand having the nucleic acid sequenceset forth in SEQ ID NO:24 and an antisense strand having the nucleicacid sequence set forth in SEQ ID NO:25; and

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:26 and second polynucleotide having the nucleic acidsequence set forth in SEQ ID NO:27.

While a preferable embodiment of the invention is directed todouble-stranded siRNA molecules wherein the two 3′ nucleotides aredeoxythymidine residues (SEQ ID Nos. 20-27), it is to be understood thatother modifications are within the scope of the present invention. Thus,the use of analogs, variants and derivatives of the sequences set forthin any one of SEQ ID NOS: 12-27 is contemplated, as long as theinhibitory activity of the SMAC/Diablo expression is retained. Forexample, in a particular embodiment, the siRNA may contain 2′-O-methyland/or phosphorothioate substituent nucleotides. According to certainexemplary embodiments, the siRNA may contain 2′-O-methyl (2′-O-ME)modification. According to these embodiments, the siRNA is selected fromthe group consisting of siRNA comprising a sense strand having thenucleic acid sequence set forth in SEQ ID NO:28 and an antisense strandhaving the nucleic acid sequence set forth in SEQ ID N0:29; siRNAcomprising a sense strand having the nucleic acid sequence set forth inSEQ ID NO:30 and an antisense strand having the nucleic acid sequenceset forth in SEQ ID N0:31; siRNA comprising a first sense strand havingthe nucleic acid sequence set forth in SEQ ID NO:32 and an antisensestrand having the nucleic acid sequence set forth in SEQ ID NO:33; siRNAcomprising a sense strand having the nucleic acid sequence set forth inSEQ ID NO:34 and second polynucleotide having the nucleic acid sequenceset forth in SEQ ID NO:35; siRNA comprising a sense strand having thenucleic acid sequence set forth in SEQ ID NO:36 and an antisense strandhaving the nucleic acid sequence set forth in SEQ ID NO:37; siRNAcomprising a sense strand having the nucleic acid sequence set forth inSEQ ID NO:38 and an antisense strand having the nucleic acid sequenceset forth in SEQ ID NO:39; siRNA comprising a first sense strand havingthe nucleic acid sequence set forth in SEQ ID NO:40 and an antisensestrand having the nucleic acid sequence set forth in SEQ ID NO:41; andsiRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:42 and second polynucleotide having the nucleic acidsequence set forth in SEQ ID NO:43.

In other particular embodiments, the siRNA is a variant, homolog orderivative of any one of SEQ ID NOs:12-27.

As used herein, the term “variant” refers to substantially similarsequences possessing common qualitative biological activities. Anoligonucleotide variant includes a pharmaceutically acceptable salt,homolog, analog, extension or fragment of a nucleotide sequence usefulfor the invention. Encompassed within the term “variant” are chemicallymodified natural and synthetic nucleotide molecules (derivatives). Alsoencompassed within the term “variant” are substitutions, additions ordeletions within the nucleotide sequence of the molecule, as long as therequired function is sufficiently maintained. Oligonucleotide variantsmay share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% sequence identity (homology). In differentembodiments, “homolog” may refer e.g. to any degree of homologydisclosed herein.

Another agent capable of silencing the expression of a SMAC/Diablo RNAis a DNAzyme molecule capable of specifically cleaving its encodingpolynucleotides. DNAzymes are single-stranded nucleic acid agents whichare capable of cleaving both single and double stranded targetsequences. A general model (the “10-23” model) for the DNAzyme has beenproposed. “10-23” DNAzymes have a catalytic domain of 15deoxyribonucleotides, flanked by two substrate-recognition domains ofseven to nine deoxyribonucleotides each. This type of DNAzyme caneffectively cleave its substrate RNA at purine:pyrimidine junctions (fora review of DNAzymes see Khachigian, L. M. 2002. Curr Opin Mol Ther 4,119-21). Examples of construction and amplification of synthetic,engineered DNAzymes recognizing single and double-stranded targetcleavage sites have been disclosed in U.S. Pat. No. 6,326,174.

Another agent capable of silencing SMAC/Diablo is a ribozyme moleculecapable of specifically cleaving its encoding polynucleotides. Ribozymesare being increasingly used for the sequence-specific inhibition of geneexpression by the cleavage of mRNAs encoding proteins of interest (Welchet al., 1998. Curr Opin Biotechnol. 9:486-96). The possibility ofdesigning ribozymes to cleave any specific target RNA has rendered themvaluable tools in both basic research and therapeutic applications. Inthe therapeutics area, ribozymes have been exploited to target viralRNAs in infectious diseases, dominant oncogenes in cancers and specificsomatic mutations in genetic disorders. Most notably, several ribozymegene therapy protocols for HIV patients are already in Phase 1 trials.More recently, ribozymes have been used for transgenic animal research,gene target validation and pathway elucidation. Several ribozymes are invarious stages of clinical trials. ANGIOZYME was the first chemicallysynthesized ribozyme to be studied in human clinical trials. ANGIOZYMEspecifically inhibits formation of the VEGF-r (Vascular EndothelialGrowth Factor receptor), a key component in the angiogenesis pathway.Ribozyme Pharmaceuticals, Inc., as well as other firms has demonstratedthe importance of anti-angiogenesis therapeutics in animal models.HEPTAZYME, a ribozyme designed to selectively destroy Hepatitis C Virus(HCV) RNA, was found effective in decreasing Hepatitis C viral RNA incell culture assays (Ribozyme Pharmaceuticals,Incorporated—http://www.rpi.com/index.html).

An additional method of silencing SMAC/Diablo is via triplex formingoligonucleotides (TFOs). In the last decade, studies have shown thatTFOs can be designed which can recognize and bind topolypurine/polypirimidine regions in double-stranded helical DNA in asequence-specific manner. Thus the DNA sequence encoding the SMAC/DiabloRNA of the present invention can be targeted thereby down-regulating theRNA molecule.

The recognition rules governing TFOs are outlined e.g. by EP Publication375408. Modification of the oligonucleotides, such as the introductionof intercalators and backbone substitutions, and optimization of bindingconditions (pH and cation concentration) have aided in overcominginherent obstacles to TFO activity such as charge repulsion andinstability, and it was recently shown that synthetic oligonucleotidescan be targeted to specific sequences (for a recent review see Seidmanand Glazer, 2003. J Clin Invest; 112:487-94).

In general, the triplex-forming oligonucleotide has the sequencecorrespondence:

oligo 3'--A G G T duplex 5'--A G C T duplex 3'--T C G AHowever, it has been shown that the A-AT and G-GC triplets have thegreatest triple helical stability. The same authors have demonstratedthat TFOs designed according to the A-AT and G-GC rule do not formnon-specific triplexes, indicating that the triplex formation is indeedsequence specific.

Thus for any given sequence in the regulatory region a triplex formingsequence may be devised. Triplex-forming oligonucleotides preferably areat least 15, more preferably 25, still more preferably 30 or morenucleotides in length, up to 50 or 100 bp.

Transfection of cells (for example, via cationic liposomes) with TFOs,and subsequent formation of the triple helical structure with the targetDNA, induces steric and functional changes, blocking transcriptioninitiation and elongation, allowing the introduction of desired sequencechanges in the endogenous DNA and results in the specific downregulationof gene expression. In addition, Vuyisich and Beal have recently shownthat sequence specific TFOs can bind to dsRNA, inhibiting activity ofdsRNA-dependent enzymes such as RNA-dependent kinases (Vuyisich andBeal, 2000. Nuc. Acids Res 28:2369-74). Additionally, TFOs designedaccording to the abovementioned principles can induce directedmutagenesis capable of effecting DNA repair, thus providing bothdownregulation and upregulation of expression of endogenous genes(Seidman and Glazer, 2003, ibid). Detailed description of the design,synthesis and administration of effective TFOs can be found in U.S.Patent Application Nos. 2003/017068; 2003/0096980; 2002/0128218 and2002/0123476 and U.S. Pat. No. 5,721,138.

It will be appreciated that nucleic acid agents capable of hybridizingSMAC/Diablo RNA may down-regulate an activity thereof by preventingSMAC/Diablo RNA binding to another downstream agent.

The inhibitory acid agents of the present invention (e.g., an siRNAmolecule such as those set forth by any one of SEQ ID NOs:12-27) can bedirectly administered to the subject or can be expressed within thesubject cell. To express such an agent (i.e., to produce an RNAmolecule) in mammalian cells, a nucleic acid sequence encoding theagents of the present invention is preferably ligated into a nucleicacid construct suitable for mammalian cell expression. Such a nucleicacid construct includes a promoter sequence and additional regulatoryelements for directing transcription of the polynucleotide sequence inthe cell in a constitutive or inducible manner. The nucleic acidconstruct may preferably include additional sequences to form anexpression vector suitable for replication and/or integration ineukaryotes, and preferably also in prokaryotes.

The type of vector may be selected e.g. for producing single-stranded ordouble-stranded RNA or DNA. Suitable vectors for producing varioussilencing oligonucleic acids are known in the art. For example, RNAiexpression vectors (also referred to as a dsRNA-encoding plasmid) arereplicable nucleic acid constructs used to express (transcribe) RNAwhich produces siRNA moieties in the cell in which the construct isexpressed. Such vectors include a transcriptional unit comprising anassembly of (1) genetic element(s) having a regulatory role in geneexpression, for example, promoters, operators, or enhancers, operativelylinked to (2) a “coding” sequence which is transcribed to produce adouble-stranded RNA (two RNA moieties that anneal in the cell to form ansiRNA, or a single hairpin RNA which can be processed to an siRNA), and(3) appropriate transcription initiation and termination sequences.

Some of these vectors have been engineered to express small hairpin RNAs(shRNAs), which get processed in vivo into siRNA-like molecules capableof carrying out gene-specific silencing. Another type of siRNAexpression vector encodes the sense and antisense siRNA strands undercontrol of separate pol III promoters. The siRNA strands from thisvector, like the shRNAs of the other vectors, have 3′ thymidinetermination signals. Silencing efficacy by both types of expressionvectors was comparable to that induced by transiently transfectingsiRNA.

Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses can be also used.

Various vectors for delivering and expressing silencing RNA moleculessuch as siRNAs are known in the art, and include for example plasmidvectors, inducible vectors, adenoviral vectors, retroviral vectors andlentiviral vectors and CMV-based vectors. Exemplary vectors includepSilencer™ vectors (Ambion), Genescript siRNA vectors, Imagenex vectors(e.g. IMG-1000, IMG-700 and IMG-1200), among others.

Various methods can be used to introduce the expression vector of thepresent invention into cells. Such methods are generally described inSambrook et al, (1989, 1992), in Ausubel et al., (1989), Chang et al.,(1995), Vega et al., (1995), and Gilboa et at. (1986), and include, forexample, stable or transient transfection, lipofection, electroporationand infection with recombinant viral vectors. In addition, see U.S. Pat.Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

Useful lipids for lipid-mediated transfer of the RNA inhibitory moleculeof the invention or construct comprising same are, for example, DOTMA,DOPE, and DC-Chol (Tonkinson et al., 1996. Cancer Investigation, 14(1):54-65). Other vectors can be used, such as cationic lipids, polylysine,and dendrimers.

Other than containing the necessary elements for the transcription ofthe inserted coding sequence, the expression construct of the presentinvention can also include sequences engineered to enhance stability,production, purification, yield or toxicity of the expressed RNA.

According to another aspect, the present invention provides an isolatedSMAC/Diablo silencing molecule selected from the group consisting of:

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:14 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:15;

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:16 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:17;

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:18 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:19;

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:22 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:23;

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:24 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:25;

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:26 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:27;

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:28 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:29;

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:30 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:31;

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:32 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:33;

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:34 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:35;

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:36 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:37;

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:38 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:39;

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:40 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:41; and

siRNA comprising a sense strand having the nucleic acid sequence setforth in SEQ ID NO:42 and an antisense strand having the nucleic acidsequence set forth in SEQ ID NO:43. Each possibility represents aseparate embodiment of the present invention.

According to certain aspects, the present invention provides a methodfor treating or delaying progression of cancer associated withover-expression of second mitochondria-derived activator ofcaspase/direct inhibitor of apoptosis-binding protein with low pI(SMAC/Diablo) in a subject in need thereof, comprising administering tothe subject an effective amount of at least one peptide conjugatecomprising a peptide derived from SMAC/Diablo-interacting protein havingan amino acid sequence at least 80% homologous to an amino acid sequenceselected from the group consisting of SEQ ID NOs:46-67, analogs,derivatives and/or fragments thereof, and a mitochondria and/or nucleustargeting moiety.

According to certain embodiments, the method comprises administering aneffective amount of a peptide conjugate comprising the amino acidsequence set forth in any one of SEQ ID NOs:69-71 and 73-74. Eachpossibility represents a separate embodiment of the present invention.

Inhibitory Peptides

The present invention discloses novel peptides derived from proteinsthat interact with SMAC/Diablo. When the peptide derived fromSMAC/Diablo-interacting protein is targeted into the cell and furtherinto the nucleus and/or the mitochondria, such that the interaction withhSMAC/Diablo occurs within the organelle(s), the interaction results ininhibition or intervention with at least one of phospholipidbiosynthesis, cell growth, cell proliferation and any combinationthereof.

According to further embodiments of the invention, the agent thatinhibits the activity of SMAC/Diablo is an inhibitory peptide targetedto the nucleus and/or to the mitochondria of a cell overexpressingSMAC/Diablo.

According to certain embodiments, the inhibiting molecule is a peptideconjugate comprising a peptide derived from SMAC/Diablo-interactingprotein having an amino acid sequence at least 80% homologous to anamino acid sequence selected from the group consisting of SEQ IDNOs:46-67, analogs, derivatives and/or fragments thereof, and amitochondria and/or nucleus targeting moiety. The mitochondria and/ornucleus targeting moiety can be peptidic or non-peptidic, and iscovalently linked to the peptide derived from SMAC/Diablo-interactingprotein directly or via linker. The targeting moiety may be linked tothe peptide derived from SMAC/Diablo-interacting protein at anyposition. According to some embodiments, the targeting moiety is linkedto the N- or the C-terminus of the peptide derived fromSMAC/Diablo-interacting protein. When the conjugate comprises acombination of mitochondria and nucleus targeting moieties each moietymay be independently linked to the N- or C-terminus of the peptide orthe mitochondria and nucleus targeting moieties can be linked in tandemto the N- or C-terminus of the peptide. According to certain exemplaryembodiments, each of the nucleus and mitochondria targeting moiety alsoenhances the permeability of the peptide conjugate through the cellmembrane.

The peptides derived from SMAC/Diablo-interacting proteins and/or thepeptide conjugate can be synthetic or recombinant.

According to certain embodiments, the nucleus and/or mitochondriatargeting moiety enhances the permeability of the peptide derived fromSMAC/Diablo-interacting protein through the nuclear or mitochondrialmembrane, respectively, and typically also through the cell membrane.

According to certain embodiments, the C-terminus of the peptideconjugate is a modified carboxy terminal group selected from the groupconsisting of an amide, ester and alcohol group. Each possibilityrepresents a separate embodiment of the present invention.

According to certain embodiments, the peptide conjugate comprises apeptide derived from hSMAC/Diablo-interacting protein having an aminoacid sequence at least 80% homologous to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:46-67, analogs, derivativesand/or fragments thereof, and a nucleus targeting moiety.

According to certain embodiments, the peptide conjugate comprises apeptide derived from hSMAC/Diablo-interacting protein having an aminoacid sequence at least 80% homologous to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:46-67, analogs, derivativesand/or fragments thereof, and a mitochondria targeting moiety.

According to some embodiments, the peptide conjugate comprises an aminoacid sequence selected from the group consisting of SEQ ID NO:69; SEQ IDNO:70; SEQ ID NO:71; SEQ ID NO:73; and SEQ ID NO:74. Each possibilityrepresents a separate embodiment of the present invention.

According to certain embodiments, the peptide conjugate furthercomprises at least one additional moiety selected from the groupconsisting of cell penetration moiety, a detectable label and a carrier.Each possibility represents a separate embodiment of the presentinvention. In some embodiments, the cell penetration moiety is a fattyacid residue. The additional moiety can be linked to the peptideconjugate directly or via a linker. Each possibility represents aseparate embodiment of the present invention.

According to some embodiments, the conjugated peptides of the presentinvention comprise a cell penetrating moiety.

In some embodiments, the amino terminus of the peptides disclosed hereinis modified. In some embodiments, the amino terminal modification isaddition of a cell penetration moiety.

In some embodiments, the carboxy terminus of the conjugated peptidesdisclosed herein is modified. In some embodiments, the carboxy terminalmodification is addition of a permeability-enhancing moiety.

Non-limitative examples of cell permeability moieties includehydrophobic moieties such as lipids, fatty acids, steroids and bulkyaromatic or aliphatic compounds. According to certain embodiments, thecell penetrating moiety is a peptide (CPP).

In some embodiments, the cell permeability moiety is covalently linkedto the N- or C-terminus of the peptide conjugate via a direct bond. Inother embodiments, the cell permeability moiety is covalently linked tothe N- or C-terminus of the peptide via a linker. In some embodiments,the cell permeability moiety is a fatty acid residue. In someembodiments, the fatty acid residue is selected from C12-C20 fattyacids. In some particular embodiments, the fatty acid residue is amyristoyl group (Myr). In additional particular embodiments, the fattyacid residue is a stearoyl group (Stear). In yet additional embodiments,the fatty acid residue is a palmitoyl group (Palm).

In some embodiments, the peptide-conjugate amino terminus is modifiedwith an amino terminal blocking group. In some embodiments, the aminoterminal blocking group is selected from the group consisting of anacetyl and alkyl. Each possibility represents a separate embodiment ofthe present invention.

In some embodiments, the carboxy terminus of the peptide conjugatesdisclosed herein is modified. In some embodiments, the carboxy terminusis modified with a carboxy terminal group. In some embodiments, thecarboxy terminal group is selected from the group consisting of amide,ester and alcohol group. Each possibility represents a separateembodiment of the present invention. In some particular embodiments, thecarboxy terminal group is an amide group.

The procedures utilized to construct peptide compounds of the presentinvention generally rely on the known principles and methods of peptidesynthesis, such as solid phase peptide synthesis, partial solid phasesynthesis, fragment condensation and classical solution synthesis.

Some of the peptides of the present invention, that do not comprisenon-coded amino acids, can be synthesized using recombinant methods knowin the art. Peptide conjugates may be synthesized chemically oralternatively may be produced recombinantly and coupled syntheticallywith the conjugating moiety.

The peptides of the invention can be used in the form ofpharmaceutically acceptable salts. As used herein the term “salts”refers to both salts of carboxyl groups and to acid addition salts ofamino or guanido groups of the peptide molecule. The term“pharmaceutically acceptable” means suitable for administration to asubject, e.g., a human. For example, the term “pharmaceuticallyacceptable” can mean approved by a regulatory agency of the Federal or astate government or listed in the U. S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. Pharmaceutically acceptable salts include those salts formedwith free amino groups such as salts derived from non-toxic inorganic ororganic acids such as acetic acid, citric acid or oxalic acid and thelike, and those salts formed with free carboxyl groups such as saltsderived from non-toxic inorganic or organic bases such as sodium,calcium, potassium, ammonium, calcium, ferric or zinc, isopropylamine,triethylamine, procaine, and the like.

Analogs and derivatives of the peptides are also within the scope of thepresent application.

“Derivatives” of the peptides of the invention as used herein coverderivatives which may be prepared from the functional groups which occuras side chains on the residues or the N- or C-terminal groups, by meansknown in the art, and are included in the invention as long as theyremain pharmaceutically acceptable, i.e., they do not destroy theactivity of the peptide, do not confer toxic properties on compositionscontaining it, and do not adversely affect the immunogenic propertiesthereof.

These derivatives may include, for example, aliphatic esters of thecarboxyl groups, amides of the carboxyl groups produced by reaction withammonia or with primary or secondary amines, N-acyl derivatives of freeamino groups of the amino acid residues, e.g., N-acetyl, formed byreaction with acyl moieties (e.g., alkanoyl or carbocyclic aroylgroups), or 0-acyl derivatives of free hydroxyl group (e.g., that ofseryl or threonyl residues) formed by reaction with acyl moieties.

“Analogs” of the peptides of the invention as used herein covercompounds which have the amino acid sequence according to the inventionexcept for one or more amino acid changes, typically, conservative aminoacid substitutions.

In some embodiments, an analog has at least about 75% identity to thesequence of the peptide of the invention, for example at least about80%, at least about 85%, at least about 90%, at least about 99% identityto the sequence of the peptide of the invention.

Conservative substitutions of amino acids as known to those skilled inthe art are within the scope of the present invention. Conservativeamino acid substitutions include replacement of one amino acid withanother having the same type of functional group or side chain e.g.aliphatic, aromatic, positively charged, negatively charged.

Conservative substitution tables providing functionally similar aminoacids are well known in the art.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K), Histidine (H);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Analogs according to the present invention may comprise alsopeptidomimetics. “Peptidomimetic” means that a peptide according to theinvention is modified in such a way that it includes at least onenon-coded residue or non-peptidic bond. Such modifications include,e.g., alkylation and more specific methylation of one or more residues,insertion of or replacement of natural amino acid by non-natural aminoacids, replacement of an amide bond with another covalent bond. Apeptidomimetic according to the present invention may optionallycomprise at least one bond which is an amide replacement bond such asurea bond, carbamate bond, sulfonamide bond, hydrazine bond, or anyother covalent bond. The design of appropriate analogs may be computerassisted. Analogs are included in the invention as long as they remainpharmaceutically acceptable and their activity is not damaged.

The inhibitory agents of the present invention can be administered to asubject per se, or in a pharmaceutical composition where they are mixedwith suitable carriers or excipients.

According to yet additional aspect, the present invention provides anisolated synthetic or recombinant peptide having an amino acid sequenceat least 80% homologous to an amino acid sequence selected from thegroup consisting of SEQ ID NOs:46-67, an analog, derivative or afragment thereof, wherein the peptide is capable of binding to humanSMAC/Diablo. According to certain embodiments, the peptide comprises theamino acid sequence set forth in any one of SEQ ID NOs:46-67. Eachpossibility represents a separate embodiment of the present invention.

According to certain embodiments, the peptide further comprises anucleus and/or mitochondria targeting moiety. The nucleus ormitochondria targeting moiety can be linked to the peptide at anyposition, typically at the N- or at the C-terminus, directly or via alinker. According to some embodiments, the conjugate further comprises acell-penetrating moiety.

According to certain embodiments, the present invention provides asynthetic or recombinant peptide having the amino acid sequence setforth in SEQ ID NO:69.

According to certain embodiments, the present invention provides asynthetic or recombinant peptide having the amino acid sequence setforth in SEQ ID NO:70.

According to certain embodiments, the present invention provides asynthetic or recombinant peptide having the amino acid sequence setforth in SEQ ID NO:71.

According to certain embodiments, the present invention provides asynthetic or recombinant peptide having the amino acid sequence setforth in SEQ ID NO:73.

According to certain embodiments, the present invention provides asynthetic or recombinant peptide having the amino acid sequence setforth in SEQ ID NO:74.

Pharmaceutical Compositions

The agents of the present invention can be administered to a subject perse, or in a pharmaceutical composition where they are mixed withsuitable carriers or excipients.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the agent accounting forsilencing the expression of SMAC/Diablo and/or modulating its activitywithin a nucleus or mitochondria of a cancerous cell overexpressingSMAC/Diablo. According to certain exemplary embodiments, the activeingredient of the invention inhibits the proliferation of cancerous celland/or reduces the growth of a tumor in cancers wherein SMAC/Diablo isoverexpressed.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,inrtaperitoneal, or intranasal injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, by intra-articularinjections or by microinjections, under arthroscopy, into theinflammatory synovial tissue (i.e. in situ).

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Preferably the pharmaceutical composition can also include DOTMA, DOPE,and DC-Chol (Tonkinson et al., 1996, ibid) as transfection agents or asadditives to transfection agents neutralizing the negative charge of thenucleotides in the RNA inhibiting molecules. A preferred example of atransfection agent is poly(ethylamine) (PEI).

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (nucleic acid agent) effective to prevent, alleviateor ameliorate symptoms of a disorder or prolong the survival of thesubject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975. In: “The PharmacologicalBasis of Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active ingredient are sufficient to induce orsuppress the biological effect (minimal effective concentration, MEC).The MEC will vary for each preparation, but can be estimated from invitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as the U.S. Food and Drug Administration(FDA) approved kit, which may contain one or more unit dosage formscontaining the active ingredient. The pack may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration. The packor dispenser may also be accommodated by a notice associated with thecontainer in a form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compositions or human orveterinary administration. Such notice, for example, may be of labelingapproved by the U.S. Food and Drug Administration for prescription drugsor of an approved product insert. Compositions comprising a preparationof the invention formulated in a compatible pharmaceutical carrier mayalso be prepared, placed in an appropriate container, and labeled fortreatment of an indicated condition, as if further detailed above.

The present invention demonstrates over-expression of SMAC/Diablo inmany types of cancer, additional to those reported in other studies(Bao, S. T., et al., 2006, ibid; Arellano-Llamas, A., et al., 2006,ibid; Yoo, N. J., et al., 2003, ibid; Shintani, M., et al., 2014 ibid;Kempkensteffen, C., et al., 2008, ibid; and Shibata, T., et al., 2007.Diagn Mol Pathol 16, 1-8). This is unexpected in view of thepro-apoptotic activity of the protein, which promotes caspase activationby binding IAPB. The present invention demonstrates that silencingSMAC/Diablo expression in various cancer cell lines over-expressingSMAC/Diablo inhibited their growth, and, furthermore, in lung cancerxenografts inhibited tumor growth and resulted in the formation ofglandular alveoli-like structures, and morphological changes indicativeof cell differentiation. Cell growth inhibition was not observed innon-cancerous cell lines, although SMAC/Diablo expression was inhibited.Inhibition of the cancer-cell growth as a result of SMAC/Diablosilencing, but not in non-cancerous cells, implies for an importantfunctional role of the protein in cancer. The results presented hereinimplies for SMAC/Diablo as an important regulator of phospholipidstransport and synthesis,

The present study has taken several approaches to elucidate themolecular mechanism underlying the effects of reducing hSMAC levels inlung A549-derived tumors, including immunoblotting,immunohistochemistry, electron microscopy, q-PCR, NGS and functionalanalysis of the data. SMAC/Diablo-silencing resulted in reduction ofKi-67, a cellular marker of proliferation, both in culture (FIG. 2K) andin tumors (FIG. 4F, G), leading to a quiescent population of cells, aswell as to a significant up-regulation of differentiation pathways(FIGS. 10A, B Tables 3-5).

Ki-67 protein is detected during all cell cycle phases, other thanresting G₀ phase. During S phase, Ki-67 protein levels markedlyincreased, with these levels being maintained in interphase and M-phase(Brunu S. et al., 1992. Cell Prolif 25, 31-40). In addition, cellstreated with si-hSMAC were arrested in S phase, reflected by inhibitionof BrdU incorporation and decreased DNA content in the si-hSMAC-TTs(FIG. 2K, L). The presence of SMAC/Diablo in the nucleus and itsfunction in phospholipids synthesis is in agreement with the observationthat during the S phase, the levels of phospholipids inside the nucleuswere reduced (Maraldi M. M., et al. 1993. J Cell Sci 107, 853-859).

SMAC/Diablo silencing in tumors resulted in a widely altered expressionof genes associated with the extracellular matrix, the cell-secretedcollagen and proteoglycan matrix overlying endothelial and epithelialcells, extracellular exosomes, and proteins in the ER and Golgi lumenassociated with vesicle formation (FIG. 10A, B, Table 3). These changesmay indicate a role for SMAC/Diablo in vesicular trafficking, exosomerelease, and extracellular matrix (ECM) deposition. Exosome cargo caninclude proteins, lipids, miRNA, mRNA, and transcription factors withthe vesicles serving as extracellular messengers, mediating cell-cellcommunication, and facilitating cancer progression and metastasis(Fujita, Y., et al., 2016. Cancer Sci 107, 385-390; Minciacchi, V. R.,et al., (2015. Semin Cell Dev Biol 40, 41-51). Exosomes released bycancer cells can also transmit signals to stromal and inflammatory cellswithin the cancer microenvironment, thus impacting the cancer ECMarchitecture and generating the cancer microenvironment. Indeed,SMAC/Diablo-silencing also affected several factors associated withstromal activity (FIG. 8, Table 3), Tumor treated with siRNA comprisingthe nucleic acid sequences set forth in SEQ ID NO:20 and SEQ ID NO:21,targeted to human SMAC/Diablo (si-hSMAC-A-TTs) showed reduced expressionof genes associated with inflammation and microenvironmenttumorigenicity, including cytokines, chemokines and their relatedreceptors, as well as intracellular proteins associated withinflammatory and immune responses (Table 5). Alterations in theexpression of these genes may be associated with reduced tumorigenicityand invasiveness.

As described hereinbelow, SMAC/Diablo silencing is also associated witha modified expression of transporters of metabolites, ions, and enzymesinvolved in metabolism (FIG. 11A, B, Table 4). The affected enzymesinclude dehydrogenases and deaminases related to cholesterol, lipid, andnucleotide synthesis, amino acid metabolism, oligosaccharidebiosynthesis, and protein glycosylation. Without wishing to be bound byany theory or mechanism of action, the inhibition of cell and tumorgrowth demonstrated herein for lung cancer cells may result frommetabolic dysregulation. In this respect, most of the affected geneswere reported to be associated with various cancers. In addition asdiscussed below, SMAC/Diablo non-apoptotic function is associated withlipid and phospholipids synthesis pathways.

Morphological analysis of A549-derived tumor sections demonstratedstructural reorganization of section derived from si-hSMAC-A-treatedtumors (si-hSMAC-A-TTs) into glandular, alveolar-like clusters. Thesestructures resembled normal lung alveoli with respect to the arrangementof endothelial cells and macrophages, as visualized by anti-CD-31 (FIG.7G) and anti-F4/80 (data not shown) antibodies staining, respectively.In contrast, the organization of endothelial cells in control sections(derived from tumors treated with non-targeted siRNA, designated hereinsi-NT-TTs) typically reflected ongoing tumor angiogenesis. Withoutwishing to be bound by any theory or mechanism of action, these findingssuggest that a reduction in the expression of SMAC/Diablo triggered AT2differentiation into AT1 cells and to morphological reorganization intolung alveoli-like structures (FIG. 7).

A549 cells are non-small cells lung carcinoma cell line, derived fromprimary tumor. A549 is characterized, as the pre-Alveolar Type IIpneumocytes (AT2) of the human lung by expression of high numbers ofmultilamellar bodies (Foster K. A. et al., 1998. Exp Cell Res, 243,359-366). While AT1 cells are flattened squamous cells accounting for˜95% of the alveolar surface and lie adjacent to capillary endothelialcells to form the pulmonary gas exchange region (FIG. 7I), AT2 cells,which cover the remaining 5% of the alveolar surface, have a compactmorphology and help clear alveolar fluid via active sodium transport.Unlike terminally differentiated and non-replicating AT1 cells, AT2cells play multiple roles and have been described as “defenders of thealveolus”. AT2 cells are the main source of cells for renewal of distallung epithelium and may either regenerate cuboidal surfactant-producingAT2 cells or trans-differentiate into AT1 cells so as to repair damage(Barkauskas, C. E., et al., 2013. J Clin Invest 123, 3025-3036) andmaintain normal lung architecture and lung elasticity (Kotton, D. N.,and Fine, A. 2008. Cell Tissue Res 331, 145-156; Rock, J. R., and Hogan,B. L. 2011. Annu Rev Cell Dev Biol 27, 493-512; Anversa, P., et al.,2011. Nature Med 17, 1038-1039). The increased expression of severalputative AT1 cell markers, including podoplanin (Barkauskas, C. E., etal., 2013, ibid), in si-hSMAC-A-TTs (FIG. 7D) supports the notion thatA549 cells undergo differentiation upon reduction of SMAC/Diabloexpression.

Further support for the differentiation of cells in the residual “tumor”in si-hSMAC-A-TTs is provided by the NGS data analysis showingup-regulation of genes associated with ion transport (FIG. 11A, Table4). Increased levels of anoctamin-1 (ANO1), a voltage-sensitivecalcium-activated chloride channel that regulates transepithelial aniontransport and essential for lung airway physiology (Rock, J. R., et al.,2009. J Biol Chem 284, 14875-14880), of the Ca²⁺-activated potassiumchannel KCNN4, and of a two-pore potassium (K2P) channel, KSNK1, whichare involved in airway surface liquid hydration (Zhao, K. Q., et al.,2012. Am J Physiol Lung Cell Mol Physiol 302, L4-L12), were noted. Thesame was also found for the epithelial Na⁺ channel (α-ENaC/SCNN1A), acritical factor during the perinatal period of lung development,involved in clearance of lung fluid (Mustafa, S. B., et al., 2014. ExpLung Res 40, 380-391). Other genes associated with lung maturation,including sodium bicarbonate cotransporters (SLC44A4, NBC), and thesodium- and chloride-dependent glycine transporter 1 (SLC6A9) weredown-regulated in si-hSMAC-A-TTs.

Without wishing to be bound by any theory or mechanism of action, thesechanges in si-hSMAC-TTs may reflect functional changes with respect toairway surface liquid hydration and the clearance associated with thematuration or/and differentiation of A549 to normal-like lung cells.

Cancer progression is associated with stromal activity, characterized byincreased deposition of collagen isoforms, laminin and fibronectin, andby heparan sulphate production, as well as of extracellular matrix (ECM)degradative enzymes and metalloproteinases (MMPs). Reduction ofSMAC/Diablo levels in tumors also altered stromal formation (FIG. 8). Asexpected for tumors, si-NT-TTs showed a thin network, dispersedthroughout the tumor and enriched with vascular formations, with bothsupporting tumor development (FIGS. 8A and 8B). On the other hand,massive fibrotic structures, resembling scar tissue, were found insi-hSMAC-A-TTs (FIG. 8A).Moreover, the decreased expression of α-SMA andgenes associated with TGF-β activity suggests reduced cancer support bythe cancer-associated-fibroblasts (CAFs). NGS results demonstrate thatmetalloproteinase involved in cancer cells invasively, as MMP2 and MMP9were reduced in si-hSMAC-A-TTs. There seemed to be no effect on collagenformation or vimentin staining in si-NT-TTs or si-hSMAC-A-TTs (data notshown), although α-SMA levels were markedly decreased in si-hSMAC-A-TTs,indicating differences in stromal output. While the myofibroblastspresent in si-NT-TTs apparently mediate processes of stromal structuresthat support tumor progression. The stromal activity in si-hSMAC-A-TTswas more reminiscent of a wound-healing process, such as a scarformation.

Immunofluorescent of staining si-NT-TTs showed the presence of SMAC inthe nucleus (FIG. 9A). Nuclear localization of SMAC/Diablo was alsodetected in the nucleus of 50% of NSCLC patient samples (FIG. 9B, 9C).

Ultrastructure analysis of si-NT-TTs and si-hSMAC-A-TTs using EM clearlydemonstrated marked changes in intracellular organelles, including thenucleus (FIG. 10D, 12). These changes included decreases inintracellular vesicles of different sizes and densities, such aslysosomes and surfactant-accumulating lamellar bodies, in si-hSMAC-A-TTs(FIG. 10D, 12A). Lamellar bodies are secretory organelles found in AT2cells that store pulmonary surfactant and are composed of 60-70% PC(Parra, E., and Perez-Gil, J. 2015. Chem Phys Lipids 185, 153-175). Asdiscussed hereinbelow, without wishing to be bound by any specifictheory or mechanism of action, the results of the present inventionsuggest that the observed morphological changes in si-SMAC-TTs areassociated with SMAC/Diablo function in phospholipid synthesis.

As demonstrated herein, the expression levels of many genes associatedwith the formation of vesicle mediating intra- and extra-cellulartransport and related to organelles, such as the ER and Golgi and toexosomes, were modified (FIG. 10A, B, 11C, Table 3-4). In addition, theexpression levels of genes encoding several enzymes associated withcholesterol and lipid transport, synthesis, degradation and regulationwere modified (FIGS. 10C, F, 11A, B). These included StAR-related lipidtransfer protein 10 (STARD10), related to lipid transfer, lipase H(LIPH), phospholipase C (PLCB4), acylglycerol-3-phosphateO-acyltransferase (AGPAT2), the elongation of very long chain fattyacids protein 4 (ELOV4) and ELOV3 and ATPase phospholipid transporting10B (ATP10B; P4-ATPase), a phospholipid flippase that can alter cellshape and which inhibits cell adhesion and spreading, and thus may beassociated with the morphological changes induced by SMAC/Diablosilencing. Moreover, analysis of phospholipids and specifically PCcontent showed a significant decrease (40-50%) in si-hSMAC-A-TTsrelative to si-NT-TTs, while phosphatidylethanolamine (PE) levels wereincreased 2-fold (FIG. 10E). PC is synthesized via two major distinctpathways, namely the triple methylation of PE byphosphatidylethanolamine N-methyltransferase (PEMT) and the de novo orcytidine diphosphate (CDP)-choline (Kennedy) pathway. The enzymesinvolved in PC biosynthesis are located at the mitochondria associatedmembranes (MAM) where the transport between ER and mitochondria takesplace. A decrease in the expression of key enzymes in the Kennedypathway, and increased PEMT expression have been demonstrated (FIG.10G). SMAC/Diablo at the mitochondrial intermembrane space (IMS) mayaffect phospholipid synthesis via the phosphatidylserine decarboxylase(PSD), an inner mitochondrial membrane enzyme facing the IMS. PSDcatalyzes the conversion of phosphatidylserine (PS) to PE with therelease of CO₂ (Jacobs R L et al. 2010. J Biol Chem 285, 22403-22413),upon which PE is converted to PC in the ER. In this respect, aconnection between mitochondrial lipid metabolism, reflected in thesynthesis of mitochondrial phosphatidylethanolamine, the differentiationprogram of breast cancer cells and loss of tumorigenicity has beenrecently presented (Keckesova, Z., et al., 2017. Nature 543, 681-686).The present invention now shows (FIG. 18) that SMAC interacts with PSDand that upon SMAC deletion, PL and PC levels were decreased 2-fold,while PE levels increased 2-fold (FIG. 10E), suggesting activation ofPSD in the absence of SMAC.

The presence of phospholipids in chromatin and the nuclear matrix andthe roles of nuclear phospholipids in the structural organization ofchromatin and nucleic acid synthesis have been demonstrated (Alessenko,A. V., and Burlakova, E. B. 2002. Bioelectrochemi 58, 13-21). Asintra-nuclear phospholipids regulate DNA replication (Maraldi et al.1993, ibid) changes seen in the expression of many genes upon silencingSMAC/Diablo expression (Tables 3-5) may result from the decrease inphospholipids levels in the cell (FIG. 10) and thus, in the nucleus.

Interestingly, SMAC/Diablo has been shown to interact with two nuclearproteins, the aryl hydrocarbon receptor nuclear translocator (ARNT),also known as the oxygen-independent β subunit of hypoxia-induciblefactors (HIF-1β) and the Mastermind-like transcriptional co-activatorMAML (Wang J et al. 2011. Mol Syst Biol 7, 536). To execute theirtranscriptional function, HIF factors must form a heterodimer between anoxygen-dependent α subunit (HIF-1α or HIF-2α) and an oxygen-independentsubunit (HIF-1β). The HIF-2α-ARNT dimer is involved in cellularadaptation to the oxygen stress related to tumor growth and progression.ARNT is also required for nuclear localization of the transcriptionalrepressors NPAS1 and NPAS3 (The C H et al., 2006. J Biol Chem 281,34617-34629). MAML2, as a transcriptional co-activator, plays animportant role in Notch signaling, regulating multiple developmentalpathways (McElhinny A S et al., 2008. Oncogene 27, 5138-5147). MAML2binds to proteins such as a cyclic AMP response element-binding protein(CREB or CBP).

Thus, as nuclear SMAC/Diablo binds HIF-1β (ARNT) and MAML2, it maycompete with HIF-1α, HIF-2α, NPAS1, or NPAS3 binding to ARNT. Similarly,by binding MAML2, SMAC/Diablo may interfere with interactions of MAML2with CBP.

Without wishing to be bound by any specific theory or mechanism ofaction, silencing the expression of SMAC/Diablo by the RNAi molecules ofthe invention, or interfering with its binding capacity to HIF-1β (ARNT)and/or MAML2 within the nucleus by the peptides of the invention,together with SMAC/Diablo-mediated regulation of phospholipid synthesisand given how nuclear phospholipids control nuclear structure andfunction, explains the effect of SMAC/Diablo on multiple signalingpathways and on proliferation of cancerous cells.

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the broad scope of the invention. One skilled inthe art can readily devise many variations and modifications of theprinciples disclosed herein without departing from the scope of theinvention.

EXAMPLES Materials and Methods Materials

The cell transfection agents JetPRIME and JetPEI were from PolyPlustransfection (Illkirch, France), SMAC/Diablo-siRNA were obtained fromGenepharma (Suzhou, China). Propidium iodide (PI), sulforhodamine B(SRB), Triton X-100, Tween-20 were obtained from Sigma (St. Louis, Mo.).Paraformaldehyde was purchased from Emsdiasum (Hatfield, Pa.).Dulbecco's modified Eagle's medium (DMEM) was obtained from Gibco (GrandIsland, N.Y.). Normal goat serum (NGS) and the supplements fetal calfserum (FCS), L-glutamine and penicillin-streptomycin were obtained fromBiological Industries (Beit Haemek, Israel). Horseradish peroxidase(HRP)-conjugated anti-mouse, anti-rabbit and anti-goat antibodies werefrom KPL (Gaithersburg, Md.). Primary antibodies, their source, and thedilutions used are detailed in Table 1 herein below. A CellTiter-Gloluciferase-based assay and TUNEL stain was obtained from Promega(Madison, Wis.). 3,3-diaminobenzidine (DAB) was obtained from(ImmPact-DAB, Burlingame, Calif.).

TABLE 1 Antibodies used Antibody Source and Cat. No. IHC WB Mousemonoclonal anti- Millipore, Billerica, —  1:10000 actin MA, MAB1501Mouse polyclonal anti- Abcam, ab8115 1:300 1:2000 SMAC/Diablo Rabbitmonoclonal anti- Abcam, ab32150 — 1:2000 pro-caspase 3 Rabbit monoclonalanti- Abcam, ab108333 — 1:1000 caspase 8 Mouse monoclonal anti- CellSignaling, 9508S — 1:1500 caspase 9 Mouse monoclonal anti- B.DBioscience, — 1:2000 Cytochrome c 556432 Rabbit polyclonal anti- Abcam,ab32516 — 1:1000 AIF Rabbit polyclonal anti- Abcam, ab137392 1:3001:2000 XIAP Rabbit monoclonal anti- Thermo Scientific, NY 1:100 — Ki67RM-9106-s1 Rat monoclonal anti- Santa Cruz 1:150 — F4/80 Biotechnology,Inc. Dallas, TX, sc52664 Rabbit polyclonal anti- Abcam, Cambridge, UK1:50  — CD31 ab28364 Rabbit polyclonal anti- Abcam, ab5694 1:200 — α-SMAMouse monoclonal anti- Abcam, ab8978 1:200 — vimentin Mouse monoclonalanti- Abcam, ab10288 1:400 — Podoplanin Rabbit polyclonal anti- Abcam,ab90716 1:250 — Prosurfactant protein C

Cell Culture and Transfection

HeLa (human cervical carcinoma) cells, A549 (non-small lung carcinoma)cells, MCF-7 (human breast carcinoma) cells, PC3 (prostate cancer)cells, HepG2 (human hepatocellular carcinoma), MDA-MB-231 (human breastcarcinoma), PANC-1 (human pancreatic carcinoma), HTB-72 (SK-MEL-28)(human melanoma), U-87MG (human glioblastoma), U-118MG (humanglioblastoma) cells and non-cancerous HaCaT (spontaneously transformedaneuploid immortal keratinocyte cell line from adult human skin),HEK-293 (human embryonic kidney), TREx-293 (human Embryonic kidney), PGA(mouse primary glia and astrocyte) cells were grown in DMEM culturemedium and H358 (non-small cell lung cancer), THP1 (human Leukemicmonocyte), KG-1α (acute myeloid leukemia) and WI38 (fibroblast derivedhuman lung) cells were grown in RPMI-1640 and EMEM culture mediumsupplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100μg/ml streptomycin, 1 mM sodium pyruvate and non-essential amino acids,and maintained in a humidified atmosphere at 37° C. with 5% CO₂.

RNA Sequences

SMAC/Diablo Human Specific siRNA used include:

si-hSMAC-A: (SEQ ID NO: 20) Sense 5′AAGCGGUGUUUCUCAGAAUUGtt3′ and(SEQ ID NO: 21) antisense 5′AACAAUUCUGAGAAACCCGCtt3′; hSMAC-B:(SEQ ID NO: 22) Sense, 5′GCAGAUCAGGCCUCUAUAAtt3′; (SEQ ID NO: 23)antisense, 5′UUAUAGAGGCCUGAUCUGCtt3′; si-hSMAC-C: (SEQ ID NO: 24) sense5′CCCGGAAAGCAGAAACCAAtt3′, (SEQ ID NO: 25) antisense,5′UUGGUUUCUGCUUUCCGGGtt3′; si-hSMAC-D: (SEQ ID NO: 26) sense5′GCUGGCAGAAGCACAGAUAtt3′, (SEQ ID NO: 27) antisense5′UAUCUGUGCUUCUGCCAGCtt3′;

si-hSMAC-A1: Sense 5′AAGCGGUGUUUCUCAGAAUUGtt3′, wherein the nucleotidesat positions 5, 10, 16 and 21 (marked in bold and underline) arederivatized by 2′-O-Me (SEQ ID NO:28) and antisense5′AACAAUUCUGAGAAACCCGCtt3′ wherein the nucleotides at positions 6, 12,and 19 (marked in bold and underline) are derivatized by 2′-O-Me (SEQ IDNO:29);

si-hSMAC-A2: Sense 5′AAGCGGUGUUUCUCAGAAUUGtt3′, wherein the nucleotidesat positions 6, 9, and 21 (marked in bold and underline) are derivatizedby 2′-O-Me (SEQ ID NO:30) and antisense 5′AACAAUUCUGAGAAACCCGCtt3′wherein the nucleotides at positions 7 and 12 (marked in bold andunderline) are derivatized by 2′-O-Me (SEQ ID NO:31);

si-hSMAC-A3: Sense 5′AAGCGGUGUUUCUCAGAAUUGtt3′, wherein the nucleotidesat positions 3, 7, 11, and 16 (marked in bold and underline) arederivatized by 2′-O-Me (SEQ ID NO:32) and antisense5′AACAAUUCUGAGAAACCCGCtt3′ wherein the nucleotides at positions 6, 10and 19 (marked in bold and underline) are derivatized by 2′-O-Me (SEQ IDNO:33);

si-hSMAC-A4: Sense 5′AAGCGGUGUUUCUCAGAAUUGtt3′, wherein the nucleotidesat positions 3, 6, 11, and 21 (marked in bold and underline) arederivatized by 2′-O-Me (SEQ ID NO:34) and antisense5′AACAAUUCUGAGAAACCCGCtt3′ wherein the nucleotides at positions 6, and19 (marked in bold and underline) are derivatized by 2′-O-Me (SEQ IDNO:35);

hSMAC-B1: Sense, 5′GCAGAUCAGGCCUCUAUAAtt3′, wherein the nucleotides atpositions 4, 9, and 15 (marked in bold and underline) are derivatized by2′-O-Me (SEQ ID NO:36); antisense, 5′UUAUAGAGGCCUGAUCUGCtt3′, whereinthe nucleotides at positions 4, 9, 15, and 18 (marked in bold andunderline) are derivatized by 2′-O-Me (SEQ ID NO:37);

hSMAC-B2: Sense, 5′GCAGAUCAGGCCUCUAUAAtt3′, wherein the nucleotides atpositions 4, 9, 13, and 17 (marked in bold and underline) arederivatized by 2′-O-Me (SEQ ID NO:38); antisense,5′UUAUAGAGGCCUGAUCUGCtt3′, wherein the nucleotides at positions 6, 12,and 18 (marked in bold and underline) are derivatized by 2′-O-Me (SEQ IDNO:39);

hSMAC-B3: Sense, 5′GCAGAUCAGGCCUCUAUAAtt3′, wherein the nucleotides atpositions 6, 10, and 15 (marked in bold and underline) are derivatizedby 2′-O-Me (SEQ ID NO:40); antisense, 5′UUAUAGAGGCCUGAUCUGCtt3′, whereinthe nucleotides at positions 2, 9, and 17 (marked in bold and underline)are derivatized by 2′-O-Me (SEQ ID NO:41);

hSMAC-B4: Sense, 5′GCAGAUCAGGCCUCUAUAAtt3′, wherein the nucleotides atpositions 4, 13, and 17 (marked in bold and underline) are derivatizedby 2′-O-Me (SEQ ID NO:42); antisense, 5′UUAUAGAGGCCUGAUCUGCtt3′, whereinthe nucleotides at positions 8, 13, and 18 (marked in bold andunderline) are derivatized by 2′-O-Me (SEQ ID NO:43);

non-targeting siRNA (si-NT): sense: 5′ GCAAACAUCCCAGAGGUAU3′ (SEQ IDNO:44) antisense: 5′ AUACCUCUGGGAUGUUUGC3′ (SEQ ID NO:45).

The siRNAs were synthesized by Genepharma (Suzhou, China). Cells wereseeded (150,000 cells/well) on 6-well culture dishes to 40-60%confluence and transfected with 10-100 nM si-NT or si-hSMAC/Diablo usingthe JetPRIME transfection reagent (Illkirch, France), according to themanufacturers' instructions.

Cells were transiently transfected with pcDNA3.1 plasmid (0.5-2 μg DNA)encoding SMAC/Diablo-GFP or pEGFP encoding for GFP using the JetPRIIVIEreagent according to the manufacturer's instructions.

Cell Death Analysis

Cells death was analyzed by propidium iodide (PI) staining and flowcytometer (Beckton-Dickinson, San Jose, Calif.) and BD CellQuest Prosoftware, or using acridine orange ethidium bromide staining (Abu-Hamad,S., et al., 2009. J Cell Sci, 122(Pt 11): p. 1906-1916).

Determination of Cellular ATP Levels

Cellular ATP levels were estimated using the luciferase-based assay(CellTiter-Glo, Promega). Cells were transfected with si-hSMAC and 48 hand 72 h post-transfection, were washed twice with PBS and seeded in96-well plates at densities of 5×10⁴ cells/ml (A549, HeLa cells andH358, non-small cell lung cancer cells). ATP levels were assayedaccording to the manufacturer's protocol and luminescence was recordedusing an Infinite M1000 plate reader.

Cell Cycle Analysis

For cell cycle analysis, cells were harvested, washed with PBS and fixedwith ice-cold 70% ethanol overnight at −20° C. The cells were thenwashed with PBS, incubated with RNase A (100 μg/ml) in PBS for 30 min at37° C. Then the cells were incubated with PI (1 μg/ml) before analysison a flow cytometer (iCyt Eclipse EC800, UK). Single cells were gatedand a minimum of 10,000 cells acquired and analyzed by FACS Eclipseanalyzer.

Sulforhodamine B (SRB) Cell Proliferation Assay

Twenty-four hours post-transfection with si-NT or si-hSMAC, cells(10,000/well) were counted and seeded in 96-well plates. Afteradditional 48, 72 or 96 hours, the cells were washed with PBS, fixedwith 10% trichloroacetic acid, and stained with SRB. SRB was extractedfrom the cells using 100 mM Tris base and absorbance at 510 nm wasdetermined using an Infinite M1000 plate reader (Tecan, Mannedorf,Switzerland).

Xenograft Experiments

Five-week-old male athymic Swiss nude mice (weight ˜19-22 g) wereobtained from Envigo and allowed a week of acclimatization to their newsurroundings. Lung cancer cells A549 (3×10⁶), or breast cancer cellsMDA-MB231 (3×10⁶) were injected subcutaneous (s.c.) into the hind legflanks of the mice. Approval for the experimental protocol was obtainedfrom the Institutional Animal Care and Use Committee of the SorokaUniversity Medical Center. Eleven days after inoculation, the developingtumors were measured in two dimensions with a digital caliper and tumorvolume was calculated as follows: volume=X²×Y/2, where X and Y are theshort and long tumor dimensions, respectively. Mice with xenograftsreaching a volume of 65-100 mm³ were randomized to receive eithernon-targeting (NT) siRNA or siRNA targeted to human hSMAC (si-hSMAC); 5animals in each group. Tumors and section thereof that received NT siRNAare designated si-NT-TTs (siRNA non-targeting treated tumors) and tumorsand section thereof that received hSMAC siRNA are designatedsi-hSMAC-TTs (siRNA hSMAC treated tumors). Treatment substances wereinjected into the established s.c. tumors using the jetPEI deliveryreagent (10 μg siRNA/20 μl jetPEI). The tumors were injected (at avolume equal to 10 to 20% of the tumor volume) with si-NT or theappropriate si-hSMAC every three days. Beginning on the day ofinoculation, mouse weight and tumor volume were monitored twice a weekusing a digital caliper. At the end of the experiment, i.e. when tumorvolume reached approximately 1300 mm³, the mice were sacrificed usingCO₂ gas, the tumors were excised and ex vivo weight was determined. Halfof each tumor was fixed in 4% buffered formaldehyde, paraffin-embeddedand processed for histological examination, while the second half wasfrozen in liquid nitrogen and stored in −80° C. for immunoblot analysis.

Quantitative Real-Time PCR (q-PCR)

Total RNA was isolated from cells using an RNeasy mini kit (Qiagen)according to the manufacturer's instructions. Complementary DNA wassynthesized from 1 μg total RNA using a Verso cDNA synthesis kit (ThermoScientific). q-PCR was performed using specific primers (KiCqStartPrimers; Sigma Aldrich) in triplicates, using Power SYBER green mastermix (Applied Biosystems, Foster City, Calif.). The levels of the targetgenes were normalized relative to β-actin mRNA levels. Samples wereamplified by a 7300 Real Time PCR System (Applied Biosystems) 95° C. for2 minutes and for 40 cycles using the following PCR parameters: 95° C.for 15 seconds, 60° C. for 1 minute, and 72° C. for 1 minute. The copynumbers for each sample were calculated by the CT-based calibratedstandard curve method. The mean fold changes (±SEM) of the threereplicates were calculated. The target genes examined and the forwardand reverse sequences of the primers used are listed in Table 2hereinbelow.

TABLE 2 Real-time PCR primers SEQ ID Gene Primers NO. β-Actin Forward  79 5′-ACTCTTCCAGCCTTCCTTCC-3′ Reverse   80 5′-TGTTGGCGTACAGGTCTTTG-3′SMAC/ Forward   81 Diablo 5′-CTGACTTCTACTTCCAGGCTGTT-3′ Reverse   825′-GCTCCTATGATCACCTGCCA-3′ XIAP Forward   83 5′-GCACGGATCTTTACTTTTGGG-3′Reverse   84 5′-GGGTCTTCACTGGGCTTC-3′ cIAP1 Forward   855′-ATCTAGTGTTCCAGTTCAGCC-3′ Reverse   86 5′-ACCACTTGGCATGTTCTACC-3′cIAP2 Forward   87 5′-CATGCCAAGTGGTTTCCAAG-3′ Reverse   885′-TCTGCATTTTCATCTCCTGGG-3′ AIF Forward   89 5′-AAGCACGCTCTAACATCTGG-3′Reverse   90 5′-TTCTCCAGCCAATCTTCCAC-3′ Cyto- Forward   91 chrome c5′-TTTGGATCCAATGGGTGATGTTGAG-3 Reverse   925′TTGAATTCCTCATTAGTAGCTTTTTTGAG-3 caspase  Forward   93 85′-GGAGCTGCTCTTCCGAATTA-3 Reverse   94 5′-GCAGGTTCATGTCATCATCC-3′caspase  Forward   95 9 5′-CTAGTTTGCCCACACCCAGT-3′ Reverse   965′-TGCTCAAAGATGTCGTCCAG-3′ caspase  Forward   97 35′-AGGACTCTAGACGGCATCCA-3′ Reverse   98 5′-TGACAGCCAGTGAGACTTGG-3′ Ki-67Forward   99 5′-CTTTGGGTGCGACTTGACG-3′ Reverse  1005′-GTCGACCCCGCTCCTTTT-3′ IL-6 Forward  1015′-CTCAATATTAGAGTCTCAACCCCCA-3′ Reverse  102 5′-AAGGCGCTTGTGGAGAAGG-3′STAT3 Forward  103 5′-GCTTTTGTCAGCGATGGAGT-3′ Reverse  1045′-TCTGCTAATGACGTTATCCAGTT-3′ NOS1 Forward  1055′-CAGTGATGATAGGATAAAGGAGGGA-3′ Reverse  106 5′-CATCATGAGCCCGTCCGC-3′SOD2 Forward  107 5′-TAAACGTAGTGTCCACGGCA-3′ Reverse  1085′-TTTCCACACGCTTATCTGCGA-3′ FOXRED2 Forward  1095′-TCAACCTCCCAAGTAGCTGG-3′ Reverse  110 5′-TTAGGAGGCCAAGACAGGTG-3′ PTGS2Forward  111 5′-CTCCTGTGCCTGATGATTGC-3′ Reverse  1125′-AACTGATGCGTGAAGTGCTG-3′ PCSK5 Forward  113 5′-TGGCACAGTCTACCGGAAAT-3′Reverse  114 5′-CCTGAGAGTGGAATGGTGGT-3′ SLC4A4 Forward  1155′-GGCTTCTTCTCTCCCACAGT-3′ Reverse  116 5′-TTCTTGGTTTGATGCCGGTG-3′ GAS6Forward  117 5′-GGAGAAGACACCACCATCCA-3′ Reverse  1185′-TCCCAGGTTGATTCAGTCCC-3′

Next-Generation Sequencing (NGS) and Functional Analysis

Libraries were prepared by the INCPM-RNA-seq unit (Weizmann Institute,Israel). Briefly, polyA fraction (mRNA) was purified from 500 ng oftotal RNA, isolated as above, followed by fragmentation and generationof double stranded cDNA. End repair, a base addition, adapter ligationand PCR amplification steps were performed according to establishedmethods. Libraries were evaluated by Qubit and TapeStation. Sequencinglibraries were constructed with barcodes to allow multiplexing of 20samples in one lane. Between 22 and 26 million reads single-end 60-bpreads were sequenced per sample on Illumina HiSeq 2500 V4 instrument.

Bioinformatics analyses were carried out at the Bioinformatics CoreFacility at the National Institute for Biotechnology in the Negev,Ben-Gurion University. Raw sequence reads were assessed for qualityusing FASTQC and further trimmed for removal of remaining adaptors andlow quality bases using Trimmomatic. The trimmed reads from each samplewere separately aligned to the human and mouse genomes (GRCh38 andGRCm38.75, respectively) using STAR v2.3.0 with default settings. Anaverage of 23.7±0.8 million reads per sample was obtained. Of these, anaverage of 13, 2.6, 7.8 and 0.3 million reads were mapped to human only,mouse only, both human and mouse, and none of them, respectively. Afterrejection of multi-mapped reads, a total of 11.5±2.5 human-specific,uniquely mapped reads, and 2.3±1.7 mouse-specific, uniquely mapped readswere subjected for further analysis. The number of aligned reads pergene per sample were counted using HTSeq count with“intersection-nonempty” (to handle reads that overlap with more than onegene) and “no stranded” as parameters. The other parameters were set totheir default. Normalization and statistical analysis for differentialexpression were carried out using DESeq2 v1.6.3. Fold change values wereconverted to linear scale, with a minus sign indicating down-regulation.

Differentially expressed genes were defined as those having a p-value<0.05, and linear fold change >1.5 and <−1.5. Functional analysis wasperformed using the Gene Ontology system, DAVID and Expander softwaretools.

Patient-Derived Samples

Chronic lymphocytic leukemia (CLL) blood samples were obtained fromSoroka University Medical Center from patients not receiving any diseasetreatment. Blood samples were also obtained from healthy volunteers.Peripheral blood mononuclear cells (PBMCs) were isolated from venousblood of CLL patients by Ficoll-Paque PLUS (GE Healthcare) densitygradient centrifugation as described previously (Admoni-Elisha, L., etal., 2016. PLoS One, 2016. 11(4): p. e0148500).

Fresh lung cancerous and non-cancerous tissue specimens were obtainedfrom the same lung cancer patients undergoing either pneumonectomy orpulmonary lobectomy to remove tumor tissue and were immediately frozenin liquid nitrogen and maintained at −80° C. until use. Cancer andnormal lung tissue surrounding the tumor were validated by hospitalpathologists. Proteins were extracted from the tissue sample using alysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1.5 mMMgCl₂, 10% glycerol, 1% Triton X-100, a protease inhibitor cocktail(Calbiochem), followed by sonication and centrifugation (10 min, 600 g).Protein concentration was determined and samples were stored in −80° C.until use.

The research was approved by the Soroka University Medical CenterAdvisory Committee on Ethics in Human Experimentation and conducted inaccordance with national laws and regulations, the ethical principlesset forth in the Declaration of Helsinki and with good clinical practiceas described in the ICH guidelines. Written informed consent wasobtained from all participants prior to entry into the study. Allsubjects received a copy of their signed and dated informed consentform.

Immunohistochemistry

Tissue microarrays (MC5003, LC807) containing cancer and normal tissuespurchased from US Biomax (US Biomax, Inc. USA) and formalin-fixed andparaffin-embedded tumor sections were immunohistochemistry (IHC)stained. Sections were deparaffinized using xylene and a graded ethanolseries. Endogenous peroxidase activity was blocked by incubating thesections in 3% H₂O₂ for 10 minutes. Antigen retrieval was performed in0.01M citrate buffer (pH 6.0) in 95° C.-98° C. for 20 minutes. Afterwashing sections with PBS containing 0.1% Triton-X100 (pH 7.4),non-specific antibody binding was reduced by incubating the sections in10% normal goat serum for 2 hr. After decanting excess serum, sectionswere incubated overnight at 4° C. with primary antibodies (sources anddilutions are detailed in Supplementary Table 1). Sections were washedwith PBST. For IHC, endogenous peroxidase activity was blocked byincubating the sections in 3% H₂O₂ for 15 min. After washing thoroughlywith PBST, the sections were incubated for 2 h with anti-mouse or antirabbit (1:250) secondary antibodies conjugated to HRP, as appropriate.Sections were washed five times in PBST and the peroxidase reaction wassubsequently visualized by incubating with 3,3-diaminobenzidine (DAB)(ImmPact-DAB, Burlingame, Calif.). After rinsing in water, the sectionswere counterstained with hematoxylin, and mounted with Vectashieldmounting medium (Vector Laboratories, Burigame, Calif.). Finally, thesections were observed under a microscope (Leica DM2500) and images werecollected at 20× magnification with the same light intensity andexposure time.

Immunoblot Staining

For immunostaining, membranes containing electro-transferred proteinsfollowing SDS-PAGE were blocked with 5% non-fat dry milk in TBS (10 mMTris 150 mM NaCl, pH 7.8) containing 0.1% Tween-20, incubated with theprimary antibodies (sources and dilutions as detailed in Table 1 or inthe corresponding example). Following washing 3 times with TBST (10 mMTris 150 mM NaCl, pH 7.8 containing 0.05% Tween-20) the membranes wereincubated with HRP-conjugated anti-mouse or anti-rabbit (1:10,000) oranti-goat (1:20,000) IgG. Enhanced EZ-ECL chemiluminescence detectionkit (Cat: 20-500-120), Biological Industries, Beit—Haemek, Ill.) wasused for detection of HRP activity. Band intensity was quantified usingFUSION-FX (Vilber Lourmat, France).

TUNEL Assay

Tumor sections were processed for the TUNEL assay using the DeadEndFluorometric TUNEL system (Promega, Madison, Wis.) according to themanufacturer's instructions. Sections were deparaffinized, equilibratedin PBS, permeabilized with proteinase K (20 μg/ml in PBS), post-fixed in4% paraformaldehyde, and incubated in TdT reaction mix (Promega) for 1 hat 37° C. in the dark. Slides were then washed in 2× saline-sodiumcitrate (SSC) buffer and counter-stained with PI (1 μg/ml), and coverslipped with Vectashield mounting medium (Vector Laboratories, Burigame,Calif.). Fluorescent images of apoptotic cells (green) and cell nuclei(red) were captured using a confocal microscope (Olympus 1X81).

Lipid Extraction

Lipids were extracted from si-NT-TTs and si-hSMAC-A-TTs as describedpreviously (Folch, J., M. et al., 1957. J Biol Chem, 226(1): p.497-509.). Briefly, 10-20 mg of tissue were added to CHCl₃/MeOH (2:1,v/v) (7.5 mg/ml) and homogenized twice using ultrasound sonication.Water (12 μl/mg) was added to the suspension; the tube was closed with aglass cap and shaken at room temperature for 2 h at 400 rpm. Thesuspension was heated to 60° C. for 10 min and subsequently stored at 8°C. for more than 1 h in order to denature proteolipids. Undissolvedmaterial was separated by filtration through a Syringe Filter (0.4 μmpore). After evaporation of solvent, the residue was re-dissolved inCHCl₃/MeOH. The suspension was filtered and the solvent was evaporatedagain. The weight of the extracted lipids was determined.

Estimation of Phospholipids, Phosphatidylcholine andPhosphatidylethanolamine

Phospholipids content of the lipids extracted from si-NT-TTs andsi-hSMAC-A-TTs as described above was determined based on the formationof a complex of the phospholipids with ammonium ferrothiocyanate asdescribed previously (Stewart, J. C., 1980. Anal Biochem, 104(1): p.10-14). The absorbance at 488 nm was determined and the amounts ofphospholipids were calculated using phosphatidylcholine (PC) a standard(10-100 μg). For phosphatidylcholine (PC) determination, samples of theextracted lipids were analyzed using ammonium thiocyanatocobaltatereagent as described previously (Yoshida, Y., E. et al., 1979. JBiochem, 86(3): p. 825-828). The absorbance at 316 nm was measured andPC amount was calculated using a phosphatidylcholine standard (10-100μg).

Phosphatidylethanolamine (PE) content of the lipids extracted fromsi-NT-TTs and si-hSMAC-A-TTs was measured using a fluorescence-basedassay as described previously (Jae-Yeon Choi et al. 2018. J. Biol. Chem293, 1493-1503). Briefly, samples of lipid extracted from tumors asdescribed above were dissolved in chloroform and after then chloroformwas evaporated under gas nitrogen. The dried extract was thenre-suspended in 0.8 mM Triton X-100. To 20 μl of lipid samples, reactionmixture including 50 μl reaction buffer (80 mM NaCl, 2 mM KPO4, pH 7.4),30 μL H₂O and 12.5 μl borate buffer (100 mM boric acid, NaCl 75 mM,sodium tetraborate 25 mM, pH, 9) were added and placed in 96 well plateand mixed well. 12.5 μl of 100 μM distyrylbenzene-bis-aldehyde (DSB-3),kindly provided by Prof. Uwe Bunz, (Organisch-Chemisches Institute,Heidelberg, Germany) prepared in 10 mM KH₂PO₄, pH 7.4 were then added.After 2 h of incubation in dark with shaking at 100rpm, fluorometricdetection of PE was carried out at λex=403 nm, λem=508 nm, using a platereader. A standard curve using purified PE was carried out in parallel.

Example 1: Expression of SMAC/Diablo in Tumors

Expression levels of SMAC/Diablo in tissue microarray slides containingsamples of randomly selected normal (10-20 healthy) controls anddifferent types of malignant cancer (40 to 80) were assessed byimmunohistochemistry (IHC) using SMAC/Diablo-specific antibodies (FIG.1A). Marked increase in SMAC/Diablo expression level was observed invarious cancer tissues, including lung, B-lymphoma, testis, colon,stomach, breast, prostate, and skin. No significant increase inSMAC/Diablo level was observed in brain, ovary, uterine, bladder,cervix, uterus, esophagus, head and neck, intestinal mucous membrane,kidney, liver, or oral cavity cancer tissues (data not shown).

Levels of SMAC/Diablo expression were higher in samples of lung cancer(non-small cell lung cancer; NSCLC), relative to samples of adjacenthealthy tissue from the same lung (FIG. 1B). Quantitative analysisshowed 3.5-fold higher levels of SMAC/Diablo expression in NSCLC tissuesamples, relative to corresponding healthy tissue (FIG. 1D).Immunoblotting analysis showed an approximately six-fold increase in theexpression of SMAC/Diablo in peripheral blood mononuclear cells (PBMCs)from chronic lymphocytic leukemia (CLL) patients, as compared to PBMCsfrom healthy donors (FIG. 1C, D).

The expression level of SMAC/Diablo in cancerous cell lines, includingHeLa, A549, H358, HepG2, MCF7, MDA-MB-231, U-87MG, U-118MG, THP1, andKG-1α cells, were about 2-4 fold higher compared to the non-cancerousTREx-293, HEK-293, HaCaT and WI-38 cell lines (FIG. 1E,F).

Example 2: Silencing of SMAC/Diablo Inhibits Cell Growth

The function of SMAC/Diablo in cancer was explored by silencing itsexpression in a number of human-derived cancer cell lines of variousorigins (i.e., HeLa, A549, H358, MCF-7, PC3, HepG2, MDA-MB-231, PANC-1,and HTB-72) using siRNA specific to human SMAC/Diablo (designatedsi-hSMAC-A, comprising a sense stand having the nucleic acid sequenceset forth in SEQ ID NO: SEQ ID NO:20 and an antisense strand having thenuclkeic acid sequnce set forth in SEQ ID NO:21). SMAC/Diablo proteinexpression levels were markedly decreased (80-90%) in all tested celllines (FIG. 2A), with a maximum of 90% decrease seen after 48 h.Ninety-six h post-transfection, the inhibition level dropped to 65%,most probably due to degradation of the siRNA or its dilution in thegrowing cells (exemplified with non-small lung carcinoma cell lines A549and H358 FIG. 2B, C and Hela Cells, FIG. 2D, E).

The effect of the SMAC/Diablo silencing on cell growth was examinedusing the sulforhodamine B (SRB) assay. In the three cell lines tested(HeLa, A549, and H358), a marked 70-80% decrease in cell proliferationwas observed 120 h post-transfection with si-hSMAC-A, whereas thecontrol transfected with non-targeting siRNA (si-NT) showed nosignificant effect on cell growth (FIG. 2F). Interestingly, inimmortalized non-cancerous cell lines, including WI38 and HaCat,si-hSMAC-A decreased SMAC/Diablo expression (FIG. 2G), yet did notaffect cell growth (FIG. 2H). Three other siRNAs designed to targethSMAC (B to D as described hereinabove) were also found to inhibitSMAC/Diablo expression and cell growth to various degrees (FIG. 2I, 2J).

si-hSMAC-A and si-hSMAC-B were modified by 2′-O methylation (2′-O Me) toform si-hSMAC-A1, si-hSMAC-A2 si-hSMAC-A3 si-hSMAC-A4 si-hSMAC-B1si-hSMAC-B2 si-hSMAC-B3 and si-hSMAC-B4 described hereinabove. Theeffect of these modified forms on SMAC/Diablo expression and on cellproliferation was examined using 30 and 50 nM of each inhibitory RNAmolecule in the lung cancer line A549. Of the examined si-hSMAC-Amodified molecules, si-hSMAC-A1 and si-hSMAC-A4 were found to be mostactive in reducing cell proliferation (FIG. 14C), and of the examinedsi-hSMAC-B modified molecules, si-hSMAC-B2 and si-hSMAC-B3 were found tobe most active (FIG. 14F).

Lung cancer cells (A549) treated with si-hSMAC-A, the most active siRNA,showed a significant decrease in the number of cells expressing the cellproliferation factor/marker Ki-67 as reflected by a decrease of about85% in Ki-67 positive cells (FIG. 2K).

As Ki-67 levels are increased with cell cycle progression, this decreasein expression suggests that cells treated with si-hSMAC-A do not advancein the cell cycle. Cell cycle analysis revealed an about 3-fold increasein the number of cells in S-phase in si-hSMAC-treated A549 cellsrelative to etoposide-treated cells (a chemotherapy medication used forthe treatments of a number of types of cancer, including lung cancer)(FIG. 2L), suggesting that SMAC/Diablo-depleted cells have decreasedcapacity to proceed in S phase.

Silencing SMAC/Diablo in HeLa, A549, and H358 cells reduced cellular ATPlevels by 20-35% (FIG. 2M), which may contribute to the observedinhibition of cell growth.

There was no significant cell death (5-10%) of HeLa, A549 or H358 cellssilenced for SMAC/Diablo (FIG. 3A-C), suggesting that the decrease incell growth was due to inhibition of cell proliferation rather thanenhanced cell death. As expected, cell death was induced by selenite.Moreover, apoptosis was induced by over-expressing SMAC/Diablo orSMAC/Diablo-GFP in HeLa, A549 and H358 cells in a concentration- andtime-dependent manner, again as expected (FIG. 3D-F).

Example 3: Silencing SMAC/Diablo Expression Inhibits Tumor Growth inMice

The effect of si-hSMAC-A was tested on a sub-cutaneous (s.c) tumorxenograft of A549 cells established in nude mice (FIG. 4). Followingtumor formation (75-90 mm³), the mice were divided into three matchedgroups, and injected every 3 days with either si-NT (group 1) orsi-hSMAC-A at 350 nM (group 2) or 700 nM (group 3). Tumor growth wasfollowed for 39 days (FIG. 4A). In animals injected with si-NT-treatedtumors (TTs), tumor volume increased by 13-fold, whereas 700 nMsi-hSMAC-A treatment reduced growth markedly (FIG. 4A). Comparing tumorsizes at the end point revealed decrease of 50% and 85% insi-hSMAC-A-TTs at the 350 nM and 700 nM levels, respectively.

All mice were sacrificed 39 days post-cell inoculation, and the tumorswere excised (FIG. 4B) and weighed (FIG. 4C). This revealed 40% and 75%decreases in tumor weight for 350 and 700 nM si-hSMAC-A-TTs,respectively, values similar to the calculated tumor volumes (FIG. 4A).Half of each tumor was next excised, fixed, and paraffin sections wereanalyzed by IHC. si-NT-TTs were strongly immunostained withanti-SMAC/Diablo antibodies. However, as expected, the staining was veryweak in si-hSMAC-A-TTs (FIG. 4D). Similar results were obtained usingqPCR (FIG. 4E). No expression of the alternative splice variantSMAC/Diablo-ε was found in the A549-derived tumors (FIG. 4E), althoughthis isoform was previously detected in healthy human tissues and inseveral cancer cell lines (Martinez-Ruiz, G. U., et al., 2014. Int JClin Exp Pathol 7, 5515-5526).

The expression levels of the cell proliferation factor Ki-67, asanalyzed by IHC staining or q-PCR, were markedly decreased (˜80%) in thesi-hSMAC-A-TTs (FIG. 4F, G), similar to the results obtained with cancercells in culture (FIG. 2K). Similar results were also obtained withMDA-MB-231 cells (FIG. 5).

Example 4: Silencing of SMAC/Diablo Alters the Expression ofApoptosis-Associated Proteins

TUNEL staining revealed a lack of significant apoptosis in eithersi-NT-TTs or si-hSMAC-A-TTs from A549-derived xenograft tumors (data notshown). As SMAC/Diablo released from mitochondria during apoptosis bindsto and counters the activities of IAPB leading to the release of boundcaspases (Verhagen, A. M., et al. 2000. Cell 102, 43-53), the expressionlevels of XIAP1, cIAP1, and cIAP2, as well as of pro-apoptotic proteinsincluding caspases 3, 8, and 9, Cyto c, and AIF were analyzed. Theexpression levels of these genes (mRNA) were noticeably decreased, asrevealed by q-PCR (FIG. 6A, B). A reduction of protein expression levelwas demonstrated by IHC for XIAP (data not shown).

In cell culture, immunoblotting (FIG. 6C,D) and q-PCR (FIG. 6E) of HeLa,A549, and H358 cells treated with si-hSMAC-A showed decrease in theexpression level not only of SMAC/Diablo but also of its bindingprotein, XIAP. SMAC/Diablo silencing also affected expression levels ofapoptosis-related proteins, including caspases 3, 8, 9, albeit in atransient effect, with maximum inhibition being noted at 48 h, followedby a lessening of the effect 96 h after transfection.

These results suggest cross-talk between the levels of expression ofSMAC/Diablo and those of a variety of apoptosis-related proteins.

Example 5: Silencing SMAC/Diablo Expression Alters Residual TumorMorphology

Hematoxylin and eosin (H&E) staining of sections from si-NT-TTs andsi-hSMAC-A-TTs lung cancer xenografts demonstrated a mostly similarmorphology of lung cancer tissue with cyst-like structures (FIG. 7A).However, further morphological analysis revealed that in si-hSMAC-A-TTs,the cells were organized in glandular-like clusters, surrounded by achain of cells (FIG. 7B), which were not seen in si-NT-TTs (FIG. 4B).These features can be interpreted as indicative of the cancer cells insi-hSMAC-A-TTs having undergone a differentiation process. Toinvestigate this point, we analyzed the expression of thepulmonary-associated surfactant protein c (SP-C) (FIG. 4C), a componentof the surface-active lipoprotein complex that is required for properbiophysical function of the lung, and found in alveolar epithelial typeII (AT2) cells (Barkauskas, C. E. et al., 2013. J Clin Invest 123,3025-3036). No significant differences in the staining of SP-C insi-NT-TTs and si-SMAC-A-TTs were found (FIG. 4C). The expression of theAT1 cells marker podoplanin (also known as T1α, PDPN), a membranalmucin-type sialoglycoprotein (Barkauskas et al., ibid), was alsoassessed (FIG. 4D, E). The higher expression levels of podoplanin insi-hSMAC-A-TTs support the suggestion that cells in the residual tumorhad undergone differentiation into AT1-like cells. (FIG. 4F)Photomicrograph of si-hSMAC-A-treated tumor stained with toluidine blue.Arrows indicate glandular-like clusters surrounded by a chain of cells.

Further structural analysis revealed that the cells organized in a chainaround the glandular-like structures were positively stained for theendothelial cell marker CD31 (FIG. 7G), and were organized in a mannerresembling lung alveoli. In contrast, in si-NT-TTs, the CD31-positivecells were flattened and randomly dispersed over the entire area of thetumor tissue (FIG. 7G). The results of the histological analysis couldreflect a scenario whereby si-NT-TTs CD31-positive cells are involved inthe process of tumor angiogenesis, while in si-hSMAC-A-TTs, the cellularorganization more closely resembles the normal physiological alveolarendothelial arrangement designed for O₂ exchange. A schematicpresentation of a cross-section through alveoli is offered forcomparison with the glandular/alveolar-like structures observed in thesi-hSMAC-A-TTs (compare FIGS. 7H and I).

Further support for this view comes from analysis of the formation ofstroma in H&E-stained si-NT-TTs and si-hSMAC-A-TTs (FIG. 8). While thestromal structures in si-NT-TTs were thin, appeared fragile, and weredispersed throughout the tissue, in si-hSMAC-TTs, massive fibroticstructures, resembling scar tissue, could be seen (FIG. 8A). Inaddition, the stromal structures in si-NT-TTs were enriched withvascular formations, associated with angiogenesis; these were barelynoticeable in si-hSMAC-A-TTs (FIG. 8B).

Staining with Sirius red and of vimentin, staining collagen andintermediate filaments, respectively, appeared to be similar insi-NT-TTs and si-hSMAC-A-TTs (data not shown). In contrast, staining forα-SMA, a myofibroblast marker, was observed mainly in si-NT-TTs, and wassignificantly reduced in si-hSMAC-A-TTs (FIG. 8C), suggesting reducedmyofibroblast infiltration.

Example 6: Over-Expressed SMAC/Diablo is Found in the Nucleus andCytosol

Interestingly, it was noticed that although SMAC/Diablo is known as amitochondrial protein, high levels were also detected in the nucleus andcytosol of NSCLC tissue microarray samples (FIG. 9). IHC analysisrevealed that SMAC/Diablo was found in the nucleus in ˜50% of thesamples (data not shown). The sub-cellular localization of SMAC/Diabloin si-NT-TTs was further analyzed by immunofluorescence staining usinganti-Cyto c antibodies as mitochondria markers and confocal microscopy(data not shown). It was found SMAC/Diablo and Cyto c were co-localizedin si-NT-TTs. Here too, SMAC/Diablo was found in addition tomitochondria also in the nuclei. As expected, no SMAC/Diablo wasdetected in si-SMAC-A-TTs.

Nuclear localization of SMAC/Diablo was also detected in lung cancertissue by IHC analysis (FIG. 9B). Analysis of various types of cancer(FIG. 1) detected nuclear localization of SMAC/Diablo, in addition toNSCLC, only in B diffuse lymphoma (Fig. data not shown).Cytosol-localized SMAC/Diablo was also found in patient-derived NSCLCtumors over-expressing SMAC (FIG. 9C).

Example 7: Next-Generation Sequencing (NGS) of si-NT-TTs andsi-hSMAC-TTs and Functional Analysis

NGS was used to investigate changes in patterns of gene expression insi-NT-TTs and si-hSMAC-A-TTs (FIG. 10, and Tables 3-5). Such analysisrevealed 848 genes, half of which are of human origin (428; 50.5%) andhalf of which are of murine origin (420; 49.5%), that displayedsignificant changes (≥1.5-fold change, adjusted P value<0.05). Assi-hSMAC is human-specific, any effect on mouse gene expression must bemediated by the human tumor cells.

Of the human genes the expression of which was modified followingSMAC/Diablo silencing, about 186 were up-regulated and 242 weredown-regulated. Functional analysis (Gene Ontology system, DAVID) ofgene expression in si-NT-TTs and si-hSMAC-A-TTs revealed differentialexpression of genes associated with key functions and pathways relatedto tumorigenicity (FIG. 10, Tables 3-5). The major functional groupswere changes were seen are presented below.

Genes associated with membranes, organelles and extracellular matrix—Theexpression of about 200 genes associated with cell membrane, exosomes,and extracellular matrix and proteins found in the lumen of the ER andGolgi were altered. While genes associated with cell membrane,extracellular exosomes and extracellular matrix were found to be bothup- and down-regulated, genes associated with ER lumen and Golgi wereonly down-regulated (FIG. 10A, B, Table 3). Some of these results werevalidated by q-PCR (FIG. 11).

Genes associated with lipid transport, synthesis andregulation—Alterations in the membranal system could reflect thatmembrane components, such those involved in phospholipid synthesis, wereimpaired upon SMAC reduction. Indeed, si-SMAC-TTs showed alterations inthe expression levels of genes associated with the transport, synthesisand regulation of lipids (FIG. 10C, Table 4). These included theelongation of long chain fatty acids protein 4 (ELOV4, 145-fold) andELOV3 (7-fold); ATP10B (25-fold) that mediates the transport ofphospholipids from the outer to the inner leaflet of various membranes;StARD10, involved in the transfer of phosphatidylcholine (PC) andphosphatidyetanolamline (PE) between membranes; LIPH lipase, catalyzingthe production of 2-acyl lysophosphatidic acid, and phospholipase C,cleaving phospholipids, that were all up-regulated. At the same time,glycerol kinase (GK), diacylglycerol kinase delta (DGKD), thatphosphorylates diacylglycerol to produce phosphatidic acid, and acyl-CoAdehydrogenase (ACAD10), that participate in the beta-oxidation of fattyacids in mitochondria, were all decreased in si-SMAC-A-TTs (FIG. 10C,Table 4). In addition, the expression of SLC44A4 that mediate cholinetransport was increased in si-SMAC-A-TTs (FIG. 11A).

Transporters of metabolites and ions and cell metabolism—Anotherinteresting group of differentially expressed genes includes thoserelated to the transport of metabolites and ions (FIG. 11A, B, Table 4).In si-hSMAC-TTs, down-regulated genes included several members of thesolute carrier family (SLC), which mediate sodium/bicarbonate,sodium/potassium/calcium co-transport, the organic anion transporter,which transports the prostaglandins PGD2, PGE1, PGE2, and themitochondrial iron transporter. Other genes down-regulated insi-hSMAC-A-TTs are associated with lung maturation, including sodiumbicarbonate co-transporters (SLC4A4, NBC), and the sodium- andchloride-dependent glycine transporter 1 (SLC6A9).

Up-regulated genes included the Ft/sucrose symporter, genes involved inthe regulation of lipid metabolism, the thiamine pyrophosphate (TPP)transporter, a cation/proton antiporter, and those encoding severalchannels for K⁺, Na⁺, Cl⁻ (FIG. 14A, Table 4). Increased levels ofanoctamin-1 (ANO1), a voltage-sensitive calcium-activated chloridechannel that regulates trans-epithelial anion transport that isessential for lung airway physiology (Rock, J. R., et al. 2009. J BiolChem 284, 14875-14880) and of the Ca²⁺-activated potassium channel KCNN4and a two-pore potassium (K2P) channel, KSNK1, which are involved inairway surface liquid hydration (Zhao, K. Q et al. 2012. Am J PhysiolLung Cell Mol Physiol 302, L4-L12), were noted. The same is true for theepithelial Na⁺ channel (α-ENaC/SCNN1A), a critical factor during theperinatal period of lung development, involved in clearance of lungfluid ((Mustafa, S. B., et al. 2014. Exp Lung Res 40, 380-391).

Metabolism-related genes whose expression was altered in si-hSMAC-A-TTsincluded dehydrogenases and deaminases responsible for mediating theconversion of oxaloacetate to phosphoenolpyruvate, alcoholdehydrogenase, hexose-6-phosphate dehydrogenase, and adenosinedeaminase, all of which were down-regulated (FIG. 11B, Table 4).Up-regulated genes encoded for enzymes involved in nucleotide synthesis(cytidine deaminase, ectonucleoside triphosphate diphosphohydrolase,calcium-activated nucleotidase 1), amino acid metabolism (peptidylarginine deiminase, glutamic pyruvate transaminase), oligosaccharidebiosynthesis, and protein glycosylation (glycosyltransferases,fucosyltransferases, beta-galactoside alpha-2,3-sialyltransferase,polypeptide N-acetylgalactosaminyltransferase) (FIG. 11B, Table 4). Mostof the transporters and metabolism-related proteins with modifiedexpression in the si-hSMAC-A-TTs have already been reported to beassociated with several cancers, in addition to the lung cancer analyzedhere (Table 4).

Inflammation and the tumor microenvironment—The si-hSMAC-A-TTs alsoshowed a reduction in the expression of genes associated withinflammation and the tumor microenvironment, including cytokines,chemokines, their related receptors, and intracellular proteinsassociated with inflammatory and immune responses (Table 5). Selectedresults were confirmed by q-PCR (FIG. 11C). These results included IL-6,known to be involved in tumorgenicity and the activation oftranscription factors, such as STAT3, associated with oncogenicity. Inaddition, the results of NGS and q-PCR analysis showed a reduction inthe levels of nitric oxide synthase 1 (NOS1), an essential factor fortumorgenicity and angiogenesis, and of mitochondrial superoxidedismutase 2 (SOD2) (Table 4, FIG. 11C) as well as genes associated withthe expression of transforming growth factor β (TGF-β) andmetalloproteinases (MMP2 and MMP9) in si-hSMAC-A-TTs (Table 5). Similarstaining of si-NT-TTs and si-hSMAC-A-TTs with the macrophage-specificantibody F4/80 was seen (data not shown).

A conclusion following from these results is that up-regulation of theexpression of SMAC/Diablo in tumor cells may be associated with anincrease in the inflammatory activity that is essential fortumorigenicity, invasiveness, metastasis, and angiogenesis.

TABLE 3 List of selected genes associated with exosome and vesicleformation differentially expressed between si-NT- and si-hSMAC-A-treatedA549 cells-derived tumors, based on NGS analysis Fold change Gene(Uniport accession) (p value) Proposed function Relation to cancerEXOSOMES CD9- (TSPAN29)  1.5 Glycoprotein/integrin belongs a prognosticbiomarker in [HGNC: 1709] (1.1 × 10⁻⁴) to tetraspanin family NSCLC CD63-(TSPAN30)  3.5 Glycoprotein/integrin belongs a prognostic biomarker intransmembrane channel- (3.88 × 10⁻¹²)  to tetraspanin family. InvolvedNSCLC like 4 [HGNC: 22998] intracellular vesicular transport processesTSPANS-tetraspanin 8  1.6 Glycoprotein/integrin belongs Ametastasis-promoting [HGNC: 11855] (2.3 × 10⁻⁵) to tetraspanin familytetraspanin TSPAN13-  1.8 GLYCOPROTEIN/INTEGRIN Overexpressed inprostate tetraspanin 13 (5.1 × 10⁻⁴) BELONGS TO cancer [HGNC: 21643]TETRASPANIN FAMILY VAMPS- vesicle-associated  1.9 V-SNARE PROTEIN,Reduces cancer invasive membrane protein 8 (1.0 × 10⁻⁵) INVOLVED INDOCKING activity [HGNC: 12647] AND/OR FUSION OF SYNAPTIC VESICLES RAB17-member RAS  1.9 A SMALL GTPASE Reduces cancer invasive oncogene family(1.0 × 10⁻⁴) Associated with epithelial activity [HGNC: 16523] polarityEPCAM- epithelial cell  2.7 Epithelial cell-cell adhesion, Poorprognosis and adhesion molecule  (3.1 × 10⁻¹¹) proliferation, migration,metastasis [HGNC: 11529] signaling and differentiation CEACAM5 (CD66e)- 2.2 Associated with adhesion and Involved in cancer carcinoembryonicantigen- (3.2 × 10⁻⁵) invasion metastasis related cell adhesion molecule5 [HGNC: 1817] CANT1- calcium activated  1.5 A nucleotidase with aOverexpressed in prostate nucleotidase 1 (4.4 × 10⁻⁴) preference for UDPcancer [HGNC: 19721] HSPG2 (Perlecan)- −1.7 A component of the vascularAssociated with lung heparan sulfate (3.5 × 10⁻⁶) extracellular matrix,involved cancer, melanoma brain proteoglycan 2 in endothelial growthmetastasis [HGNC: 5273] FSTL1-follistatin-like 1 −1.8 Involved in thelung inhibits the invasion and [HGNC: 3972] (7.6 × 10⁻⁸) developmentmetastasis CD274- PD-L1 molecule −1.9 Involved in the suppression ofAssociated with tumoral [HGNC: 17635] (3.3 × 10⁻⁴) immune responseimmune escape NID2- nidogen 2 −2.1 A basal lamina protein Tumorsuppressor (osteonidogen) (1.8 × 10⁻⁶) [HGNC: 13389] GAS6- growtharrest- −1.7 AXL receptor tyrosine kinase Overexpressed in lung specific6 [HGNC: 4168] (3.0 × 10⁻³) activation cancer GPIBA-glycoprotein Ib −1.7A receptor for von Willebrandt Associated with cachexia (platelet),alpha (1.0 × 10⁻³) factor (VWF) in metastatic melanoma polypeptide[HGNC: 4439] IGFBP7- insulin-like −1.8 It is involved in IGF high It isassociated with growth factor binding (2.1 × 10⁻⁵) affinity interactionand cell NSCLC progression protein 7 [HGNC: 5476] adhesion EXTRACELLULARSPACE CCL24- chemokine (C-C  1.9 CHEMOTACTIC FOR Associated with poormotif) ligand 24 (3.0 × 10⁻⁴) RESTING T prognosis in hepatocellular[HGNC: 10623] LYMPHOCYTES AND carcinoma (HCC) and EOSINOPHILS colorectalcancer CCL28- chemokine (C-C  1.9 Involved in inflammatory Supportcancer metastasis motif) ligand 28 (8.8 × 10⁻⁴) response [HGNC: 17700]SPINK1- serine peptidase  1.9 A serine protease inhibitor Associatedwith prostate inhibitor, Kazal type 1 (4.4 × 10⁻⁴) cancer [HGNC: 11244]PROC- protein C  1.8 involved in hemostasis, The expression is(inactivator of coagulation (1.2 × 10⁻³) inflammation and signalassociated with an factors Va and Villa) transduction improved lungcancer [HGNC: 9451] prognosis SPINT1- serine peptidase  1.7 A serineprotease inhibitor Associated with lung inhibitor, Kunitz type 1 (6.8 ×10⁻⁵) cancer malignant pleural [HGNC: 11246] effusion LGALS3- lectin, 1.6 Associated with cell adhesion, Involved in A549 cells,galactoside-binding, (1.6 × 10⁻⁴) cell activation and promoted CSCformation. soluble, 3 [HGNC: 6563] chemoattraction, cell growth Involvedin involved in and differentiation, cell cycle, inflammation andfibrosis and apoptosis CX3CL1-chemokine (C-  1.6 Activates integrins andelicits Associated with cancer X3-C motif) ligand 1 (1.6 × 10⁻⁴)adhesive and migratory progression [HGNC: 10647] leukocytes functionsIGFBP1- insulin-like −1.6 Improves and inhibits IGF a tumor growthinhibitor in growth factor binding (8.3 × 10⁻⁶) interaction with cellssurface breast cancer cells protein 1 [HGNC: 5469] receptors MST1-macrophage −1.6 Hepatocyte growth factor-like Antiproliferative activityin stimulating 1 (hepatocyte (1.9 × 10⁻³) protein A549 cells growthfactor-like) [HGNC: 7380] CPM- carboxypeptidase M −1.7 Presents inpneumocytes and Associated with poor [HGNC: 2311] (1.0 × 10⁻⁴) involvedin EGFR activation prognosis in lung adenocarcinomas WISP2- WNT1inducible −1.7 The CCN family of proteins Promotes a stem-like cellsignaling pathway protein 2 (9.1 × 10⁻⁶) involved in cell adhesion,phenotype in breast cancer [HGNC: 12770] migration, proliferation,cells. Acts as a tumor differentiation suppressor in colorectal cancerIGFBP3- insulin-like −1.8 blocks the access of IGF-1, 2 Functions as atumor growth factor binding (4.5 × 10⁻⁸) to IGF-1R suppressor protein 3[HGNC: 5472] CELL MEMBRANE CLDN4- claudin 4  2.4 Involved in tightjunction- Associated with a good [HGNC: 2046] (3.9 × 10⁻⁹) specificobliteration of the prognosis in NSCLC, SCC intercellular space.subgroups EMP1- epithelial  1.8 Involved in tight junction- Reducedexpression membrane protein 1 (4.7 × 10⁻⁵) specific obliteration of theassociated with lung cancer [HGNC: 3333] intercellular space.progression BAMBI- BMP and activin  1.7 Negatively regulates TGF-betaInvolved in the suppression membrane-bound inhibitor (2.9 × 10⁻⁴)signaling of invasiveness [HGNC: 30251] CDH3- cadherin 3, type 1,  1.7Cadherin protein related to Associated with expression P-cadherin(placental) (4.0 × 10⁻³) stem cells properties in lung cancer [HGNC:1762] DSC2- desmocollin 2  1.6 Cadhcrin-type protein involved Reducedexpression [HGNC: 3036] (1.4 × 10⁻⁵) in adjacent cells interactionassociated with poor prognosis in pancreas, colorectal and esophagealsquamous cell cancers TNS1- tensin 1 −1.6 Actin-binding protein,Involved in EMT, tumor [HGNC: 11973] (7.7 × 10⁻⁶) promotes motilityinvasion and metastasis CLMP- Coxsackie- and −1.7 Involved in cell-celladhesion Associated with poor adenovirus receptor (9.1 × 10⁻⁴) prognosis(CXADR)-like membrane protein [HGNC: 24039] C5AR1 (CD88)- −1.7Complement component 5a Associated with poor complement component 5a(4.0 × 10⁻³) receptor involved in prognosis in NSCLC receptor 1 [HGNC:1338] inflammatory response. CNTNAP2-contactin −1.8 The protein ofneurexin family, Associated with poor associated protein-like 2 (9.4 ×10⁻⁵) involved in cell adhesion. prognosis in breast cancer [HGNC:13830] ITGA1 (CD49a)- integrin −1.9 Involved in cell-cell adhesion,Involved in lung subunit alpha 1 (1.1 × 10⁻⁵) inflammation and fibrosislymphangioleiomyomatosis (LAM) invasiveness ACKR3- atypical −2.2 areceptor for chemokines Increased expression in chemokine receptor 3(1.8 × 10⁻⁵) CXCL11 and CXCL12/SDF1 lung SCC [HGNC: 23692] EXTRACELLULARMATRIX COL17A1 (BP180)-  2.1 involved in epidermal adhesionUnderexpression in breast collagen, type XVII, alpha (3.0 × 10⁻⁷) cancerassociated with poor 1 [HGNC: 2194] prognosis COL5A1- collagen, type V,−1.5 Belongs to type I collagen and ASSOCIATED WITH A alpha 1 [HGNC:2209] (5.0 × 10⁻⁴) binds to DNA, heparan sulfate, RENAL CANCER POORthrombospondin, heparin, and PROGNOSIS, GASTRIC, insulin BREAST CANCERSCOL12A1- collagen, type −1.5 Interacts with type I collagen Associatedwith drug XII, alpha 1 [HGNC: 2188] (9.8 × 10⁻⁴) and the surroundingmatrix resistance in ovarian cancer cell lines, breast cancerprogression, colorectal cancer metastasis VCAN- versican −1.5 Involvedin cells-extracellular, Associated with an [HGNC: 2464] (1.3 × 10⁻³)matrix interaction advanced disease SMOC1- SPARC related −1.6 Amatricellular protein Increased in brain cancers. modular calciumbinding 1 (3.7 × 10⁻⁴) interferes with growth factor Involved inangiogenesis. [HGNC: 20318] receptor signaling Interacts with NID2 VTN-vitronectin −1.6 A cell adhesion and spreading Not correlated withdisease [HGNC: 12724] (1.0 × 10⁻²) factor progression COL4A2- collagen,type −1.8 The major structural Associated with lung IV, alpha 2 [HGNC:2203] (1.1 × 10⁻⁶) component of glomerular cancer, melanoma brainbasement membranes, fonning metastasis, gastric cancer a ‘chicken-wire’meshwork together widi laminins, proteoglycans and entactin/nidogenFBN1- fibrillin 1 −1.7 Extracellular matrix Associated with an ovarian[HGNC: 3603] (2.0 × 10⁻³) glycoprotein serves as a cancer progressstructural component BMPER- BMP binding −1.7 Inhibitor of BMP activityAssociated with tumor endothelial regulator (4.2 × 10⁻³) growth. [HGNC:24154] BMP2-bone morphogenetic −1.8 Implicated in the developmentAssociated with tumor protein 2 [HGNC: 1069] (5.2 × 10⁻⁴) of bone andcartilage. Involved growth. in the hedgehog pathway, TGF beta signalingpathway activation GPC6- glypican 6 −1.8 extracellular matrix proteins,Involved in invasion of [HGNC: 4454] (9.6 × 10⁻⁵) breast cancer NLGN2-neuroligin 2 −1.8 Involved in cell-cell No direct association [HGNC:14290] (1.4 × 10⁻⁶) interactions via its interactions reported withneurexin family members (see CNTNAP2) ADAMTS9- ADAM −2.2Metalloproteinase and involved Inhibits angiogenesis andmetallopeptidase with (2.1 × 10⁻⁵) in the transport from the tumorgrowth in ESCC and thrombospondin type 1 endoplasmic reticulum to theNPC motif, 9 [HGNC: 13202] Golgi apparatus COL4A1- collagen, type −2.2The major structural Associated with lung IV, alpha 1 [HGNC: 2202] (9.6× 10⁻⁷) component of glomerular cancer, melanoma brain basementmembranes (GBM), metastasis, gastric cancer forming a ‘chicken-wire’meshwork together with laminins, proteoglycans and entactin/nidogenABI3BP (TARSH)- ABI −2.4 The cellular senescence-related Decreasedexpression is family, member 3 (NESH) (1.1 × 10⁻⁶) gene associated withlung cancer binding protein progression and metastasis [HGNC: 17265]EDIL3 (Del1)-EGF-like −2.5 Interacts with the αv/β3 ASSOCIATED WITHrepeats and discoidin I-like (1.6 × 10⁻⁶) integrin receptor and promotesLUNG CANCER CELL domains 3 [HGNC: 3173] adhesion of endothelial cells.PROLIFERATION AND INVASION CCBE1- collagen and −2.7 Involved inangiogenesis A prognostic marker for calcium binding EGF (4.3 × 10⁻⁷)process gastrointestinal stromal domains 1 [HGNC: 29426] tumorprogression TGFBI-Transforming −1.9 Involved in cell-collagen Associatedwith invasion, growth factor-beta-induced (1.5 × 10⁻⁵) interactions andcells adhesion signaling and ECM protein ig-h3 interaction in different[HGNC: 11771] types of cancer MMP2-Metalloproteinase 2 −2.9 Involved inangiogenesis, Associated with cancer (72 kDa type IV (6.9 × 10⁻⁶) tissuerepair, tumor invasion, progression by ECM collagenase) [HGNC: 7166]inflammation, and degradation in different adierosclerotic plaquerupture types of tumor and degrading extracellular matrix proteinsENDOPLASMIC AND GOLGI LUMENS AGR3- anterior gradient 3  4.1 Required forcalcium-mediated Associated with ovarian [HGNC: 24167] (1.33 × 10⁻⁶) regulation (ER) cancer RYR1- ryanodine receptor  2.5 Ca²⁺ channel thatmediates the Associated with breast 1 [HGNC: 10483] (5.6 × 10⁻³) releaseof Ca²⁺ from the cancer [HGNC: 22998] sarcoplasmic reticulum into thecytoplasm (ER/SR) NCK2- NCK adaptor  2.0 Adapter protein whichAssociated with ovarian protein 2 [HGNC: 7665] (2.04 × 10⁻⁶)  associateswith tyrosine- cancer phosphorylated growth factor receptors or theircellular substrates (ER) SEC61G- Sec61 gamma  1.8 Protein translocationin the Associated with pediatric subunit [HGNC: 18277] (4.3 × 10⁻⁴)(ER). Ependymomas BACE1- beta-site APP- −1.5 Amyloid precursor proteinMay be involved in tumor cleaving enzyme 1 (2.2 × 10⁻³) (APP) proteaseangiogenesis [HGNC: 933] FOXRED2- FAD- −1.5 Functions in endoplasmicFOXRED2 is a dependent oxidoreductase (2.0 × 10⁻³) reticulum associatedtranscriptomic fingerprint domain containing 2 degradation of KRAS[HGNC: 26264] H6PD (GDH)- hexose-6- −1.6 Oxidizes hexose-6-phosphatesAssociated with breast phosphate dehydrogenase (1.3 × 10⁻⁴) and glucosecancer poor prognosis (glucose 1-dehydrogenase) [HGNC: 4795] ACAD10-acyl-CoA −1.7 Acyl-CoA dehydrogenase only No direct associationdehydrogenase family, (1.6 × 10⁻³) active with R- and S-2-methyl-reported member 10 [HGNC: 21597] C15-CoA (mitochondria) TM4SF20-transmembrane −1.7 Polytopic transmembrane No direct association 4 L sixfamily member 20 (1.26 × 10⁻⁸)  protein that inhibits reported [HGNC:26230] intramembrane proteolysis (RIP) of CREB3L1 (ER)TMEM147-AS-TMEM147 −1.7 Antisense to transmembrane No direct associationantisense RNA 1 (8.5 × 10⁻³) protein (ER) reported [HGNC: 51273] AHCYL2-−1.7 Regulating the No direct association adenosylhomocysteinase- (4.05× 10⁻⁴)  sodium/bicarbonate reported like 2 [HGNC: 22204] cotransporterSLC4A4 activity and Mg²⁺⁻sensitivity (ER) ITPR1-inositol 1,4,5- −1.7Intracellular channel mediating Associated with renal trisphosphatereceptor, ripe (7.9 × 10⁻⁴) Ca²⁺ release from the ER upon cancer 1[HGNC: 6180] stimulation by inositol 1,4,5- trisphosphate (ER) CHERP-calcium −1.7 Involved in Ca²⁺ homeostasis, Associated with homeostasisendoplasmic (7.05 × 10⁻⁴)  growth and proliferation neuroblastomareticulum protein (ER) [HGNC: 16930] PTGS2 (COX-2)- −1.8 Convertsarachidonate to Involved in hunor grow prostaglandin-endoperoxide (1.4 ×10⁻⁴) prostaglandin H2 and metastasis synthase 2 [HGNC: 9605] P4HA3-prolyl 4- −1.9 Involved in the post- Associated with breast hydroxylase,alpha (5.5 × 10⁻⁵) translational formation of 4- cancer progressionpolypeptide III hydroxy proline in -Xaa-Pro- [HGNC: 30135] Gly-sequences in collagens PPAP2B- phosphatidic −1.8 Catalyzes theconversion of Associated with non small acid phosphatase type 2B (1.31 ×10⁻⁶)  phosphatidic acid (PA) to cells lung carcinoma [HGNC: 9229]diacylglycerol and hydrolyzes lysophosphatidic acid (ER) CYP26B1-cytochrome −1.8 Involved in the metabolism of Associated with esophagealP450, family 26, subfamily (3.85 × 10⁻⁴)  retinoic acid (RA), (ER)squamous cell carcinoma. B, polypeptide 1 [HGNC: 20581] CLGN- calmegin−1.9 A chaperone for a range of Associated with breast [HGNC: 2060] (8.6× 10⁻⁴) proteins (ER) cancer PCSK5- proprotein −2.3 Endoproteaseactivity protein Underexpressed in lung convertase subtilisin/kexin (2.7× 10⁻⁶) cancer type 5 [HGNC: 8747] COX10-AS1- COX10 −2.1 Antisense to(ER) COX10 No direct association antisense RNA 1 (7.12 × 10⁻³)  protein,which converts reported [HGNC: 38873] protoheme IX and famesyldiphosphate to heme O. ABCC6- ATP-binding −2.3 Transports glutathioneAssociated with pancreatic cassette, sub-family C (1.4 × 10⁻³)conjugates as leukotriene-c4 carcinoma (CFTR/MRP), member 6 (LTC4) andN-ethylmaleimide [HGNC: 57] S-glutathione (ER). RIC3- RIC3 acetylcholine−2.7 Promotes functional expression No direct association receptorchaperone (1.6 × 10⁻³) of homomeric alpha-7 and reported [HGNC: 30338]alpha-8 nicotinic acetylcholine receptors at the cell surface (ER,Golgi) UGT1A7- UDP −3.5 UDPGT is important in the Associated with lungglucuronosyltransferase 1 (4.6 × 10⁻³) conjugation and subsequent cancerfamily, polypeptide A7 elimination of potentially toxic [HGNC: 12539]xenobiotics and endogenous compounds. This isoform has specificity forphenols (ER) CEMIP- cell migration −3.6 Mediates depolymerization ofAssociated with colorectal inducing protein, (1.06 × 10⁻⁸)  hyaluronicacid (HA) via the cancer hyaluronan binding cell membrane-associated[HGNC: 29213] clathrin-coated pit endocytic pathway (ER) FAM20A- familywith −4.4 An allosteric activator of the Associated with cancer sequencesimilarity 20, (4.8 × 10⁻³) Golgi serine/threonine protein stem cellsmember A [HGNC: 23015] kinase FAM20C (ER, Golgi) UGT1A9-UDP −17.2  UDPGTis important in the Associated with glucuronosyltransferase 1 (9.8 ×10⁻⁴) conjugation and subsequent gastrointestinal cancer family,polypeptide A9 elimination of potentially toxic [Source: HGNCxenobiotics and endogenous Symbol; Acc: HGNC: 12541] compounds. Thisisoform has specificity for phenols (ER) ER AND MITOCHONDRIA KLK6-kallikrein-related  25.83 Serine protease (ER, Associated with NSCLCpeptidase 6 [HGNC: 6367] (9.83 × 10⁻⁵)  mitochondria) SGK1- −1.7Serine/threonine-protein kinase Associated with NSCLCserum/glucocorticoid (7.2 × 10⁻⁸) regulating a wide variety of ionregulated kinase 1 channels, transporters, cellular [HGNC: 10810]enzymes, transcription factors (ER, mitochondria) Mitochondria BIK-BCL2-interacting  2.1 Accelerates apoptosis by Associated with lungkiller (apoptosis-inducing) (2.8 × 10⁻³) binding to anti- apoptosiscancer [HGNC: 1051] proteins Bcl-X(L), BHRF1 or Bcl-2 suppresses thisdeath- promoting activity (mitochondria) ENDOG- endonuclease G  1.8Involved in replication of Associated with lung [HGNC: 3346] (2.09 ×10−³)  mitochondrial DNA, as cancer AIF1 released from mitochondria inthe time of apoptosis and is function, as endonuclease in Nucleus(mitochondria). CHCHD7- coiled-coil-  1.6 Unknown No direct associationhelix-coiled-coil-helix (1.48 × 10⁻³)  (mitochondria) reported domaincontaining 7 [HGNC: 28314] MYO19- mitochondrially −1.6 Actin-based motorAssociated with encoded NADH (4.33 × 10⁻⁵)  molecule with ATPase gliomadehydrogenase 5 activity localized to the [HGNC: 7461] mitochondrionouter membrane, involved in the interaction of mitochondria withcytoskeleton (mitochondria) MT-ND5- mitochondrially −1.6 Subunit of theIt may be associated encoded NADH (2.65 × 10⁻⁴)  mitochondrialrespiratory with breast cancer dehydrogenase 5 chain NADH [HGNC: 7461]dehydrogenase (Complex I) ANXA10- annexin A10 −1.7 Ca²⁺ -dependentAssociated with lung [HGNC: 534] (9.0 × 10⁻³) phospholipid bindingcancer (Mitochondria) CYP24A1- cytochrome −1.9 Plays a role in It may beassociated P450, family 24, subfamily (1.15 × 10⁻⁸)  maintaining Ca²⁺with lung cancer A, polypeptide 1 homeostasis. [HGNC: 2602](Mitochondria) MT-ND4L- −1.9 Subunit of the Associated withmitochondrially encoded (3.36 × 10⁻⁶)  mitochondrial respiratorychildhood acute NADH dehydrogenase 4L chain NADH lymphoblastic [HGNC:7460] dehydrogenase (Complex leukemia I) (Mitochondria) C10orf2 (TWINK)-−1.9 Involved in mitochondrial No direct association chromosome 10 open(6.5 × 10⁻⁴) DNA (mtDNA) reported reading frame 2 metabolism. Could[HGNC: 1160] function as an adenine nucleotide-dependent DNA helicase(mitochondria) STARD13- StAR-related −2.2 GTPase-activating protein Itis known, as a tumor lipid transfer (START) (5.39 × 10⁻⁵)  for RhoA, andfor Cdc42. suppressor in lung domain containing 13 Involved in lipidcancer [HGNC: 19164] binding(mitochondria) CPS1- carbamoyl- −4.8Involved in the urea cycle Associated with lung phosphate synthase 1, (7.9 × 10⁻¹⁸) of ureotelic animals, cancer mitochondrial removingexcess [HGNC: 2323] ammonia from the cell (mitochondria)

TABLE 4 Transporters and metabolism related genes differentiallyexpressed between si-NT- and si-hSMAC-A-treated A549 cells derived lungtumors Fold change Proposed function & Gene (Uniport accession) (pvalue) cellular localization Relation to cancer TRANSPORTERS SLC4A4-solute carrier family −3.3  Regulates bicarbonate Plays a role in growth4 (sodium bicarbonate (3.1 × 10⁸)  influx/efflux at the basolateral andmigration of colon cotransporter), member 4 membrane of cells and andbreast cancer. [HGNC: 11030] intracellular pH. (Cell membrane) SLCO2B1-solute carrier −2.8  Mediates the Na+-independent Associated withprostate organic anion transporter (4.1 × 10¹⁰) transport of organicanions, cancer. family, member 2B1 taurocholate, the prostaglandins[HGNC: 10962] PGD2, PGE1, PGE2, leukotriene C4, thromboxane B2. (Cellmembrane) SLC6A9- solute carrier family −1.7  Play a role in glycinetransport Associated with Thyroid 6 (neurotransmitter transporter, (5.7× 10⁻⁴) and thus in glycine regulation of cancer glycine), member 9 NMDAreceptor-mediated [HGNC: 11056] neurotransmission. (Cell membrane)SLC24A1- solute carrier family −1.6  Controlling the calcium No directassociation 24 (sodium/ potassium/calcium  (1 × 10⁻³) concentration ofouter segments reported exchanger), member 1 during light and darknessand [HGNC: 10975] plays a key role in the process of light adaptation.(Cell membrane) SLC25A37- solute carrier −1.5  Mitochondrial irontransporter Associated with prostate family 25 (mitochondrial iron (1.2× 10⁻⁴) mediates iron uptake in cancer transporter), member 37developing erythroid cells and [HGNC: 29786] playing a role in hemebiosynthesis, (IMM) KCNK1- potassium channel, 1.7 Contributes to passiveOverexpressed in lung subfamily K, member 1 (1.1 × 10⁻⁷) transmembranepotassium cancer [HGNC: 6272] transport and regulation of the restingmembrane potential. (Cell membrane, recycling endosome and cytoplasmicvesicle) SLC45A3- solute carrier family 1.8 H+/sucrose symporter slc45a3as Up-regulated by 45, member 3 [HGNC: 8642] (9.4 × 10⁻⁴) an osmolytetransporter in the androgens and play a kidney, regulation of lipid rolein prostate cancer metabolism in oligodendrocytes. malignancy (Cellmembrane) KCNN4- potassium channel 1.8 Required for maximal calciumOverexpressed in ovarian calcium-activated channel, (6.8 × 10⁻⁵) influxand proliferation during cancer subfamily N, member 4 the reactivationof naive T-cells. [HGNC: 6293] (Cell membrane) GLTP- glycolipid transfer1.8 Catalyzes the transfer of various Overexpressed in oral protein[HGNC: 24867] (1.4 × 10⁻³) glycosphingolipids between squamous cellcarcinoma membranes SCNN1A- sodium channel, 1.9 Plays an essential rolein Prognostic marker for non-voltage-gated 1 alpha (4.2 × 10⁻⁵)electrolyte and blood pressure pulmonary subunit [HGNC: 10599]homeostasis and taste perception. adenocarcinoma (Apical cell membrane)SLC44A4- solute carrier family 2.2 Choline and Thiamine Associated withprostate 44, member 4 [HGNC: 13941] (2.6 × 10⁻⁶) pyrophosphate (TPP)transporter and pancreatic cancers (Cell membrane) TMC8- transmembrane2.4 Play a role as ion channel. Involved in EV-HPV channel-like 8 [HGNC:20474] (5.8 × 10⁻⁷) (Endoplasmic reticulum oncogenicity and membrane)squamous cell carcinoma SLC9A3R2- solute carrier 2.5 Cation/protonantiporter and Associated widi breast family 9, subfamily A (NHE3, (6.3× 10⁻⁷) scaffold protein in the plasma cancer cation proton antiporter3), membrane. Acts as scaffold member 3 regulator 2 protein in thenucleus. [HGNC: 11076] (Endomembrane, nucleus) CRACR2A- calcium release3.2 Acts as a cytoplasmic calcium- No direct association activatedchannel regulator 2A  (1 × 10⁻⁴) sensor and plays a key role in reported[HGNC: 28657] store-operated calcium entry in T-cells by regulating CRACchannel activation. (Cytoplasm) ANO1-anoctamin 1, calcium 3.4 Atransepithelial anion transport Overexpressed in activated chloridechannel (3.4 × 10⁻⁵) and smooth muscle contraction. different ty pes ofcancer. [HGNC: 21625] (Cell membrane, Cytoplasm) TMC4- transmembrane 6.3Play a role as ion channels. No direct association channel-like 4 [HGNC:22998]  (6.2 × 10⁻¹²) (Membrane) reported ATP10B- ATPase, class V, 25.5 Catalyzes ATP-dependent Overexpressed type 10B [HGNC: 13543] (7.8 ×10⁻⁵) transport of aminophospholipids in lung adenocarcinoma and ensuresthe maintenance of and peripheral blood asymmetric distribution of cellsphospholipids (cytoplasmic vesicles and ER membrane) METABOLISM RELATEDENZYMES PCK1- −6.9  Catalyzes the conversion of Low level expressionphosphoenolpyruvatecarboxykinase  (2.3 × 10⁻¹¹) oxaloacetate toassociated with 1 [HGNC: 8724] phosphoenolpyruvate. hepatocarcinogenesis(Cytoplasm) ADH4 - alcohol dehydrogenase −2.6  Catalyzes the reaction:Downregulated in 4 (class II) (4.2 × 10⁻⁴) An alcohol + NAD+ → anovarian and liver cancer [HGNC: 252] aldehyde or ketone + NADH.(Cytoplasm) ADARB1- adenosine −2.1  Hydrolytic deamination of Associatedwith deaminase, RNA-specific, B1 (8.1 × 10⁻⁵) adenosine to inosine.(Nucleus) numerous carcinomas [HGNC: 226] and sarcomas ACAD 10- acyl-CoA−1.7 Associated with fatty acid beta Associated w ith reduceddehydrogenase family, member (1.6 × 10⁻³) oxidation. (Mitochondria)viability in melanoma 10, [HGNC: 21597] and pancreatic cancer cellsH6PD - hexose-6-phosphate −1.6  Associated with glucose Associated withdehydrogenase [HGNC: 4795] (1.2 × 10⁻⁴) metabolic process. (Endoplasmicchemoresistance activity reticulum) CAD- carbamoyl-phosphate −1.6 Catalyzes the reaction 2 ATP + Associated with sy nthetase 2, aspartate(1.3 × 10⁻⁴) L-glutamine + HCO3— + H2O → colorectal, transcarbamylase,and 2 ADP + phosphate + L- prostate cancers dihydroorotase [HGNC: 1424]glutamate + carbamoyl phosphate. (Cytoplasm and nucleus) ACSL4-acyl-CoAsynthetase −1.6  Activation of long-chain fatty Associated with breast,long-chain family member 4 (1.0 × 10⁻²) acids for both synthesis ofcolon, brain and [HGNC: 3571] cellular lipids, and degradation lungcancers via beta-oxidation DGKD-diacylglycerol kinase, −1.5  Involved in1,2-diacyl-sn- Associated with delta 130 kDa [HGNC: 2851] (2.7 × 10⁻⁴)glvcerol phosphorylation prostate cancer FASN-fatty acid synthase −1.5 Catalyzes the formation of long- Associated with [HGNC: 3594] (3.3 ×10⁻⁴) chain fatty acids from acetyl- lung cancer and others CoA,malonyl-CoA and NADPH ORMDL2-sphingolipid 1.5 Negative regulator of Nodirect association biosynthesis regulator 2 (4.0 × 10⁻³) sphingolipidsynthesis reported [HGNC: 16037] CANT1- calcium activated 1.5Nucleotidase involved in UDP, Associated with nucleotidase 1 [HGNC:19721] (4.0 × 10⁻⁴) GDP, UTP, GTP melanoma dephosphorylation. (Golgi,Endoplasmatic reticulum) CMAS- cytidine 1.5 Involved in the pathway N-Associated with breast monophosphate N- (7.4 × 10⁻⁴) acetylneuraminatemetabolism. cancer acetylneuraminic acid (Nucleus) synthetase [HGNC:18290] GPT2- glutamic pyruvate 1.5 Formation of pyruvate and Associatedwith colon, transaminase (alanine (1.4 × 10⁻³) glutamate bytransamination liver cancers. aminotransferase) 2 between alanine and 2-[HGNC: 18062] oxoglutarate. (Mitochondria) MGAT4B- mannosyl (alpha- 1.5Glycosyltransferase involved in Associated with mouse 1,3-)-glycoprotein beta-1,4-N- (6.7 × 10⁻⁴) transfer GlcNAc to the corecarcinogen-induced acetylglucosaminyltransferase, mannose residues.(Golgi) hepatocarcinoma. isozyme B [HGNC: 7048] ST3GAL4- ST3 beta- 1.5Involved in protein Associated with gastric galactoside alpha-2,3- (4.5× 10⁻³) glycosylation. (Golgi) carcinoma. sialyltransferase 4 [HGNC:10864] AGPAT2- l-acylglycerol-3- 1.6 Converts lysophosphatidic acidUpregulated in breast phosphate O-acyltransferase 2 (8.4 × 10⁻⁵) (LPA)into phosphatidic acid. and cervical cancer. [HGNC: 325] (Endoplasmicreticulum membrane) GALNT5- 1.6 Involved in protein Associated withpolypeptide N- (1.7 × 10⁻⁵) glycosylation. (Golgi) hepatoblastoma.acetylgalactosaminyltransferase 5 [HGNC: 4127] GALNT12- 1.6 Involved inprotein Associated with breast, polypeptide N- (3.3 × 10⁻⁴)glycosylation. (Golgi) colorectal cancers.acetylgalactosaminyltransferase 12 [HGNC: 19877] ST6GALNAC1- (alpha-N-1.6 Involved in protein Associated with acetyl-neuraminy1-2,3-beta- (6.2× 10⁻⁴) glycosylation. (Golgi) esophageal squamous galactosyl-1,3)-N-cell carcinoma and acetylgalactosaminide alpha- prostate cancer.2,6-sialyltransferase 1 [HGNC: 23614] PLCB4 - phospholipase C, beta 1.7Involved in inositol phosphate Biomarker for uveal 4 [HGNC: 9059] (9.2 ×10⁻⁴) metabolic process. (Cytosol, melanoma. Nucleus, smooth Endoplasmicreticulum) BLVRA - biliverdin reductase 1.7 Oxidation of bilirubin usingOverexpressed in liver A [HGNC: 1062] (2.1 × 10⁻³) NAD(P) to biliverdin.cancer. (Cytoplasm) GCNT3- glucosaminvl (N- 1.7 Involved in carbohydrateAssociated with acetyl) transferase 3, mucin (1.0 × 10⁻²) metabolicprocess. (Golgi) pancreatic, colon cancer. type [HGNC: 4205] ADI1 -acireductone 1.8 Formation of formate and 2-keto- Downregulated indioxygenase 1 [HGNC: 30576] (1.8 × 10⁻⁴) 4-mediyldiiobutyrate. (Cytosol,prostate cancer. Nucleus and Cell membrane) FUT6- 1.8 Involved inL-fucose catabolic Associated with Fucosyl transferase 6 (alpha (1.5 ×10⁻³) process attenuation of EGFR (1,3) fucosyltransferase) (Golgi)signaling and invasivity. [HGNC: 4017] LIPH - lipase, member 1.9Hydrolyzes specifically Overexpressed in lung [HHGNC: 18483] (3.1 ×10⁻³) phosphatidic acid (PA) to and breast cancer. produce 2-acyllysophosphatidic acid (LPA) and fatty acid. (Membrane) GALNT6- 1.9Involved in O-glycan processing Associated with polypeptide N-  (1.8 ×10−⁴) (Golgi) pancreatic, breast acetylgalactosaminyl cancers.transferase 6 [HGNC: 4128] CDA - cytidine deaminase 2.0 Cytidinedeaminase. (Cytosol, Overexpressed in cancer [HGNC: 1712] (1.1 × 10⁻³)extracellular region) FAXDC2- fatty acid 2.1 Involved in fatty acidReduced expression is hydroxy lase domain containing (5.7 × 10⁻⁵)biosynthetic process. associated with acute 2 [HGNC: 1334](Endoplasmatic reticulum) myeloid leukemia and acute megakaryoblasticleukemia. B3GNT6- UDP-GlcNAc: 2.1 Involved in Associated with prostatebetaGal beta-1,3-N- (1.6 × 10⁻⁴) glucuronosyltransferase activity.cancer. acetylglucosaminyltransferase 6 (Golgi) (core 3 synthase) [HGNC:24141] ENTPD8 - ectonucleoside 2.7 Involved in purine and May beinvolved in the triphosphate (2.1 × 10⁻³) pyrimidine metabolism. (Cellchemical formation of diphosphohydrolase 8 membrane) DNA adducts. [HGNC:24860] FUT3- fucosyltransferase 3 2.7 Involved in oligosaccharideAssociated with (galactoside 3(4)-L- (2.9 × 10⁻⁷) biosynthetic process.(Golgi) metastasis and survival fucosyltransferase, Lewis blood rategroup) [HGNC: 4014] CYB5R2 -cytochrome b5 2.8 Involved in desaturationand Act as a tumor reductase 2 [HGNC: 24376]  (3 × 10⁻⁴) elongation offatty acids, suppressor in cholesterol biosynthesis, drug nasopharyngealmetabolism. (Cytosol, Nucleus, carcinoma smooth ER) DHRS9 -dehydrogenase/ 2.8 Involved in retinol metabolic Downregulated inreductase (SDR family) (5.6 × 10⁻⁴) process, colorectal cancer member 9[HGNC: 16888] (Endoplasmic reticulum) SDR16C5- short chain 3.0 Involvedin the oxidation of No direct association dehydrogenase/reductase family(1.1 × 10⁻⁴) retinol to retinaldehyde. reported 16C, member 5(Endoplasmic reticulum) [HGNC: 30311] STARD10 - StAR-related lipid 3.7Involved in phospholipid Associated with breast transfer (START) domain (1.1 × 10⁻¹³) transport. (Cytosol, Nucleus) cancer containing 10 [HGNC:10666] PADI1- peptidyl arginine 3.8 Deamination of arginine Involve intumor deiminase, type I (2.1 × 10⁻³) residues. progression [HGNC: 18367](Cytosol, Extracellular exosome) FAR2- fatty acyl CoA 4.1 Catalyzes thereduction of fatty No direct association reductase 2 [HGNC: 25531] (1.5× 10⁻⁵) acyl-CoA to fatty alcohols reported DGAT2- diacylglycerol O- 4.9Catalyzes the terminal step in Associated with acyltransferase 2 (2.5 ×10⁻⁴) triacylglycerol synthesis using Hepatocarcinoma [HGNC: 16940]diacylglycerol and fatty acyl CoA ELOVL3-fatty acid elongase 3 7.4Catalyzes the long-chain fatty No direct association [HGNC: 18047] (1.0× 10⁻³) acids elongation toward C18 reported acyl-CoAs ELOVL4 fatty acidelongase 4 145.2  Catalyzes the long-chain fatty Hypermethylated in[HGNC: 18047]  (6.2 × 10⁻¹⁷) acids elongation, elongates C24:0 pancreas,colorectal and C26:0 acyl-CoAs carcinoma REDOX STATE REGULATIONNOS1-nitric oxide synthase, −5.9  Produces nitric oxide (NO), Abundantin low- neuronal [HGNC: 7872] (1.6 × 10⁻⁶) which is a ROS,neurotransmitter differentiated, breast and angiogenesis regulatorgynaecological, central nerve system cancer and NSCLC SOD2- superoxidedismutase −1.9  Inactivates ROS, as superoxide Increased expression in[Mn], mitochondrial (3.7 × 10⁻⁵) anion radicals prostate, lung and colon[HGNC: 11180] cancer GSTP1- glutathione S- −1.6  Conjugates reducedglutathione Associated with lung transferase pi 1 [HGNC: 4638] (2.4 ×10⁻³) to a wide number of exogenous cancer and endogenous hydrophobicelectrophiles (mitochondria) TXN-Thioredoxin 2.1 Involved in redoxreactions Prognostic biomarker in [Source: HGNC (1.8 × 10⁻⁴) through thereversible oxidation NSCLC and breast Symbol; Acc: HGNC: 12435] ofdithiol to a disulfide cancer PXDN-peroxidasin homolog 3.3 Participatein H₂O₂ metabolism Involved in melanoma [HGNC: 14966] (5.1 × 10⁻⁸) andperoxidative reactions invasion. The decreased expression is observed inmyeloid leukaemia

TABLE 5 List of selected genes associated with Inflammationdifferentially expressed between si-NT- and si-hSMAC-A-treated A549cells-derived tumors, NGS analysis Fold change Proposed function & Gene(Uniport accession) (p value) cellular localization Relation to cancerInflammatory ligands CCL24- chemokine (C-C motif)  1.9 CHEMOTACTIC FORAssociated with poor ligand 24 [HGNC: 10623] (3.0 × 10⁻⁴) RESTING Tprognosis in HCC and LYMPHOCYTES AND colorectal cancer EOSINOPHILSCCL28- chemokine (C-C motif)  1.9 Involved in inflammatory Supportcancer metastasis ligand 28 [HGNC: 17700] (8.8 × 10⁻⁴) response PROC-protein C (inactivator of  1.8 involved in hemostasis, The expression iscoagulation factors Va and (1.2 × 10⁻³) inflammation and signalassociated with an VIIIa) [HGNC: 9451] transduction improved lung cancerprognosis CX3CL1-chemokine (C-X3-C  1.6 Activates integrins andAssociated with cancer motif) ligand 1 [HGNC: 10647] (1.6 × 10⁻⁴)elicits adhesive and progression migratory leukocytes functions TNFAIP6(TSG6)- −1.6 Interacts with ECM proteins Associated with poor tumornecrosis factor, alpha- (2.2 × 10⁻³) during inflammation and prognosisinduced protein 6 tumorigenesis [HGNC: 11898] C5- complement component 5−1.7 Involved in late complement Associated with a [HGNC: 1331] (1.0 ×10⁻²) components formation C5- favorable C9 and inflammationmicroenvironment for progress cancer progression GAS6- growtharrest-specific 6 −1.7 AXL receptor tyrosine kinase Overexpressed inlung [HGNC: 4168] (3.0 × 10⁻³) activation cancer TNFSF14 (LIGHT)- −1.8Involved as a costimulatory TNFSF14 is involved in tumor necrosis factor(1.8 × 10⁻³) factor in anticancer anticancer immune- superfamily, member14 lymphocytes cytotoxic stimulatory activity [HGNC: 11930] activityCD274-(PD-L1) molecule −1.9 A costimulatory molecule, Associated with amore [HGNC: 17635] (3.3 × 10⁻⁴) involved in the inhibition of aggressivensclc T-cells proliferation and cytokine production IL6- interleukin 6−2.5 Involved in inflammatory Over-expressed in [HGNC: 6018] (1.5 ×10⁻⁶) response NSCLC RECEPTORS AND CO-RECEPTORS IL1R1- interleukin 1receptor, −1.6 Receptor for IL1A, IL1B and Associated with breast type I[HGNC: 5993] (4.0 × 10⁻⁴) IL1RN, mediates NF-kappa- cancer cellsinvasivity B and MAPK activation IL18BP- interleukin 18 binding −1.6Interacts with IL-18, inhibits Produced in the ovarian protein [HGNC:5987] (1.0 × 10⁻²) its activity and Th1 immune cancer microenvironmentresponse to regulate immune response IL27RA- interleukin 27 −1.6Receptor for IL27, acts Resulting in the receptor, alpha [HGNC: 17290](1.0 × 10⁻²) through STAT3 and STAT1 promotion of tumor growth OSMR-oncostatin M receptor −1.7 Binds IL31 together with Associated with poor[HGNC: 8507] (6.7 × 10⁻⁶) IL31RA to activate STAT3 prognosis in cervicalsquamous cell carcinoma TLR3- toll-like receptor 3 −1.7 Anucleotide-sensing TLR Associated with lung [HGNC: 11849] (5.0 × 10⁻³)activated by double-stranded metastasis by neutrophil RNA, leading toNF-kappa-B activation activation MRC2 (ENDO180)- mannose −1.7 Involvedin the remodeling Associated with prostate, receptor, C type 2 (3.6 ×10⁻³) of ECM cooperating with the breast cancers and [HGNC: 16875] MMPsmetastasis C5AR1 (CD88)-complement −1.7 Receptor for the chemotacticAssociated with poor component 5a receptor 1 (4.0 × 10⁻³) andinflammatory peptide prognosis [HGNC: 1338] anaphylatoxin C5aLBP-lipopolysaccharide binding −2.1 Binds to LPS and promotes It was notchanged in lung protein [HGNC: 6517] (1.2 × 10⁻⁴) the cytokines releaseadenocarcinoma ACKR3- atypical chemokine −2.2 a receptor for chemokinesIncreased expression in receptor 3 [HGNC: 23692] (1.8 × 10⁻⁵) CXCL11 andCXCL12/SDF1 lung SCC CYTOSOLIC MOLECULES CREB3L3- cAMP responsive 2.0Transcription factor No direct association element binding protein3-like 3 (4.3 × 10⁻⁴) activates expression of acute reported [HGNC:18855] phase response (APR) genes in inflammatory response IRF6-interferon regulatory 1.6 DNA-binding transcriptional Involved in tumorfactor 6 [HGNC: 6121] (1.0 × 10⁻³) activator suppression and SCCsdifferentiation. PTGS2 (Cox-2)- prostaglandin - −1.8 Convertsarachidonate to Involved in tumor grow endoperoxide synthase 2 (1.4 ×10⁻⁴) prostaglandin H2 and metastasis (prostaglandin G/H synthase andcyclooxygenase) [HGNC: 96051

Example 8: Ultrastructure and Lipid Synthesis in si-NT-TTs andsi-hSMAC-A-TTs

As SMAC silencing resulted in morphological changes (FIG. 7), thesub-cellular ultrastructure of si-NT-TTs and si-hSMAC-A-TTs wereanalyzed using transmission electron microscopy (TEM) (FIGS. 10D, 12A).In si-NT-TTs, a massive amount of intracellular vesicles of differentsizes and densities, such as lysosomes and large vesicles containingsurfactant-accumulating lamellar bodies were seen. Such vesicles werenot observed in the tumors where hSMAC/Diablo was silenced(si-hSMAC-A-TTs) (FIG. 10D, 12A).

In addition, in si-NT-TTs, the major DNA form in the nucleus is darklystaining heterochromatin, while in si-SMAC-TTs, the DNA is mainly foundas euchromatin and not readily stained (FIGS. 10D, 12A, B).Interestingly, euchromatin is prevalent in cells that are active in thetranscription of many genes, while heterochromatin is most abundant incells that are less active. This may be associated with celldifferentiation processes. Moreover, the sizes of nuclei insi-hSMAC-A-TTs are almost twice those in si-NT-TTs and showed lowerchromatin staining (FIG. 10D, 12A, B). The large nuclei in si-NT-TTscould result from membrane fluidity and/or osmolality changes, asreflected in the modified expression of transporters (FIG. 11A, Table4). Changes in the expression of genes associated with cell membrane,exosomes, and ER- and Golgi-related proteins (FIG. 11A, B) and in cellultrastructure (FIG. 10D, E), are summarized in a schematic model (FIG.12C).

The amounts of total phospholipids and PC in si-NC-TTs and si-SMAC-A-TTslipid extracts were analyzed (FIG. 10F). Total phospholipids and PC weredecreased by 47% and 37%, respectively, in si-SMAC-A-TTs.

These results are in agreement with the findings that SMAC/Diablosilencing resulted in alterations in the expression levels of genesassociated with the transport, synthesis and regulation of lipids (FIG.10C, Table 4). Therefore, using q-PCR, the levels of genes encodingenzymes associated with diacylglycerol (DAG) synthesis, such as glycerolkinase (GK), and the mitochondrial proteins glycerol-3-phosphateacyltransferase 1 and 4 (GPAM1, GPAM4) were analyzed. All were found tobe decreased in si-SMAC-TTs (FIG. 10F). In addition, levels of mRNAencoding enzymes involved in PC synthesis were also decreased (FIG.10F), whereas those encoding other enzymes associated with PC synthesisfrom phosphatidylethanolamine, namely phosphatidylethanolamineN-methyltransferase (PEMT), and choline/ethanolamine kinase A (CHKA),cholinephosphotransferase 1 (CHPT1) and phospholipase A2 (PLA2G1B), wereincreased. These results are presented in the PC and DAG synthesispathways depicted in FIG. 10G.

Example 9: SMAC/Diablo Interacting Peptides

A network of proteins interacting with SMAC/Diablo (FIG. 15A) wasidentified using Thebiogrid database for protein-protein interactions(thebiogrid.org), based on the following methods to define interactions:Two hybrid analysis;

-   -   Affinity capture-Western blots;    -   Affinity capture-MS;    -   Co-crystal structure    -   Biochemical activity    -   Protein-peptide interaction;    -   Förster resonance energy transfer

Fourteen (14) proteins were selected as being associated withSMAC/Diablo, all of human (Homo sapiens) origin:

-   1. XIAP: X-linked inhibitor of apoptosis Protein (NP_001158.2;    Uniprot-P98170)-   2. HTRA2: HtrA serine peptidase 2 Protein (NP_037379.1;    Uniprot-O43464)-   3. BIRC2: Baculoviral IAP repeat containing 2 Protein (NP_001157.1;    Uniprot-Q13490)-   4. CD40: Tumor necrosis factor receptor superfamily member 5    (NP_001241.1; Uniprot-P25942)-   5. TRAF2: TNF receptor associated factor 2 Protein (NP_066961.2;    Uniprot-Q12933)-   6. UBE2K: Ubiquitin conjugating enzyme E2 K Protein (NP_005330.1;    Uniprot-P61086)-   7. NR4A1: Nuclear receptor subfamily 4 group A member 1 Protein    (NP_001189163.1; Uniprot-P22736)-   8. MAML2: Mastermind like transcriptional coactivator 2 Protein    (NP_115803.1; Uniprot-Q8IZL2)-   9. ARNT: Aryl hydrocarbon receptor nuclear translocator isoform 1    Protein (NP_001659.1; Uniprot-P27540)-   10. BIRC5: Baculoviral TAP repeat containing 5 Protein (NP_001159.2;    Uniprot-O15392)-   11. AREL1: Apoptosis resistant E3 ubiquitin protein ligase 1 Protein    (NP_001034568.1; Uniprot-O15033)-   12. GFER: Growth factor, augmenter of liver regeneration Protein    (NP_005253.3; Uniprot-P55789)-   13. MTFR1: Mitochondrial fission regulator 1 Protein (NP_055452.3;    Uniprot-Q15390)-   14. GMPPB: GDP-mannose pyrophosphorylase B Protein (NP_037466.2;    Uniprot-Q9Y5P6).

A custom glass-bound peptide array consisting of 768 overlappingpeptides (of 20-25 amino acid length) derived from the above-selectedproteins was prepared by Intavis, Bioanalytical instruments, Kolen,Germany.

The peptide array was incubated overnight at 4° C. with purified SMAC(0.8 μM) and then blotted with anti-SMAC antibodies (1:2000) andincubated for additional 4 h at 4° C. followed by 2 h incubation withHRP-conjugated anti-mouse IgG and detection using the EZ-ECLchemiluminescence detection kit as described hereinabove.

Dark spots, marked with their location on the peptide array, representbinding of SMAC to peptides derived from the SMAC-interacting proteins(FIG. 15B). The peptide origin, name and SEQ ID NOs. are presented inTable 6 below.

TABLE 6 Peptides derived from proteins associated withSMAC/Diablo that interacted with SMAC/Diablo SMAC/ Peptide Diablodesignation inter- (Spot SEQ acting location ID protein FIG. 15)Peptide Sequence NO. BIRC5 1H13 SGCAFLSVKKQFEELTLGEFLKLD 46 UBE2K 2C18VRFITKIWHPNISSVTGAICLDILK 47 BIRC2 1G12 LIRKNRMALFQQLTCVLPILDNLLK 481G13 FQQLTCVLPILDNLLKANVINKQEH 49 IE23 SASLGSTSKNTSPMRNSFAHSLSPT 50 1G18LVKGNAAANIFKNCLKEIDSTLYKN 51 1G21 FVDKNMKYIPTEDVSGLSLEEQLRRL 52 TRAF21I23 MAAASVTPPGSLELLQPGFSKTLLGTK 53 1J20 DGCGKKKIPREKFQDHVKTCGKCRV 541K16 AGRIPAIFSPAFYTSRYGYKMCLRI 55 1K20 HLSLFFVVMKGPNDALLRWPFNQKV 56MAML2 2D9 GAGLLGGGSVTPRVHSAIVERLRAR 57 2G8 QQQQQQQPSSQPAQSLPSQPLLRS* 582G9 SSQPAQSLPSQPLLRSPLPLQQKLLL 59 ARNT 2I14 MAATTANPEMTSDVPSLGPAIASGN 602J5 VSHMKSLRGTGNTSTDGSYKPSFLT 61 2J14 DVDKLREQLSTSENALTGRILDLKT 62 NR4A11M17 GDNASCQHYGVRTCEGCKGFFKRTV 63 1N22 ASCLKEHVAAVAGEPQPASCLSRLL 64 1N23AVAGEPQPASCLSRLLGKLPELRTL 65 CD40 1I13 LVVQQAGTNKTDVVCGPQDRLRALV 66HTRA2 1C9 ALGGIRWGRRPRLTPDLRALLTSGT 67 *Letter marked in italicrepresent overlapping sequence within SEQ ID NO: 58 and SEQ ID NO: 59.

Example 10: SMAC/Diablo Specific Interaction with Peptides Derived fromSMAC/Diablo-Interacting Proteins

The effect of peptide 2C18 (SEQ ID NO:47) derived from the SMAC/Diablointeracting proteins, Ubiquitin conjugating enzyme E2 K Protein (UBEK2);and of peptide 1G12 (SEQ ID NO:48) derived from Baculoviral IAP repeatcontaining 2 Protein (BIRC2) on SMAC/Diablo capability to interact withthese and other peptides was examined.

The glass-bound peptide array described hereinabove was used.SMAC/Diablo (0.8 μM) was incubated with each of the peptides—2C18 (2.4μM) and 1G12 (2.4 μM) for 2 hr at 24° C. and then incubated with thepeptide array for 4 h at 4° C. Then the slides were washed andSMAC/Diablo antibodies (1:2000) were added, followed by incubation withHRP-conjugated anti-mouse IgG after which chemiluminescent spots weredetected. Corresponding glass-bound peptide array incubated overnightwith 0.8 μM free SMAC/Diablo, blotted with the same concentration ofSMAC/Diablo antibodies followed by incubation with HRP-conjugatedanti-mouse IgG served as a control. As expected, pre-incubation ofSMAC/Diablo with peptide 2C18 or peptide 1G12 significantly decreased oreven prevented the interaction of SMAC/Diablo with the correspondingpeptides, as the binding site is already occupied by the free peptidepre-incubated with SMAC/Diablo. This is indicated by the decreasedintensity or disappearance of chemiluminescence in the peptide spotposition compared to the control (FIG. 16 and FIG. 17, respectively).Moreover, pre-incubation of SMAC/Diablo with peptide 2C18 decreasedSMAC/Diablo interaction with the array-bound peptides 1M17 and 1N23derived from Nuclear receptor subfamily 4 group A member 1 Protein(NR4A1) and peptide 1K20 derived from TNF receptor associated factor 2Protein (TRAF2) (FIG. 16B, eliminated spots are circled). Pre-incubationof SMAC/Diablo with peptide 1G12 significantly reduced SMAC/Diablointeraction with 1G12 and also the interaction with peptide 1G13, alsoderived from BIRC2 and having overlapping sequence with peptide 1G12,but also with peptide 1H13, derived from Baculoviral IAP repeatcontaining 5 Protein (BIRCS) and peptide 2D9, derived from Mastermindlike transcriptional coactivator 2 Protein (MAML2) (FIG. 17B, eliminatedspots are circled).

Example 11: Cell Growth Inhibition by Peptides of the Invention

Considering the presence of the overexpressed SMAC/Diablo in themitochondria intermembrane space and in the nucleus it is important totarget SMAC/Diablo interacting peptide to these compartments,facilitating the cellular uptake and nucleus and/or mitochondrialocalization of the peptides of the present invention is of significantimportance for the therapeutic use of the peptides of the invention.Several conjugates of peptides of the invention fused to cell- andnucleus or mitochondria penetrating peptide were designed (Table 7below).

The tetrapeptide Arg-D-Arg-Arg-Lys (Cindy A P et al. 2010. Bioorganic &Medicinal Chemistry 18:3564-3569) was used to facilitate cellular uptakeand nuclear localization. The peptide is designated herein cell/nucleuspenetrating peptide (nuCPP, SEQ ID NO:71).

The tetrapeptide D-Arg-Dmt-Orn-Phe, wherein Dmt=2,6-dimethyl-L-tyrosine,(Carmine Pasquale Cerrato, et al. 2015. The FASEB Journal, 29(11):4589-4599), was used to facilitate cellular uptake and mitochondrialocalization. The peptide is designated herein cell/mitochondriapenetrating peptide (mtCPP, SEQ ID NO:72)

A peptide comprising transferrin-receptor binding domain (Tf) having thesequence His-Ala-Ile-Tyr-Pro-Arg (SEQ ID NO:80 and a peptide comprisingthe Drosophila antennapedia (ANTP) domain having the sequenceMet-Arg-Gln-Ile-Lys Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys (SEQ IDNO:81) each was used to facilitate uptake of the peptide through thecell plasma membrane and into the cytosol.

TABLE 7 Examined peptide conjugates Conjugate description Targeted toSEQ ID NO. 1H13-nuCPP Nucleus 69 1H13-mtCPP-NH₂ Mitochondria 731G12-nuCPP Nucleus 70 nuCPP-1G12 Nucleus 71 mtCPP-2C18 Mitochondria 74ANTP-2C18 Cytosol 75 TF-1G12 Cytosol 76

A549 cells were seeded in 96-well plate (5000 cells/well). After 24 hcells were incubated with each of the examined peptides at aconcentration of 5, 10, or 30 μM for 48 h in a serum (10%) containingmedium or for 24 h in a serum-free medium. Then, cells were washed withPBS, fixed with 10% trichloroacetic acid, and analyzed for cell growthusing the SRB method described herein above. Cell growth with noaddition of a peptide was taken as a control (100% cell growth). Table 8presents the cell growth in the presence of the peptide as % of control.Results are the mean±SD (n=3).

TABLE 8 Effect of cells treatment with peptides on cell growth Cellpenetrating Concentration Cell growth, peptide Targeted to μM % ofcontrol Control 0 100 1H13-nuCPP Nucleus 5 92 ± 0.018 1H13-nuCPP Nucleus10 57 ± 0.013 1H13-nuCPP Nucleus 30 30 ± 0.009 1H13-mtCPP-NH₂Mitochondria 5 85 ± 0.022 1H13-mtCPP-NH₂ Mitochondria 10 60 ± 0.0181H13-mtCPP-NH₂ Mitochondria 30 49 ± 0.014 1G12-nuCPP Nucleus 5 109 ±0.013  1G12-nuCPP Nucleus 10 76 ± 0.024 1G12-nuCPP Nucleus 30 39 ± 0.013nuCPP-1G12 Nucleus 5 96 ± 0.012 nuCPP-1G12 Nucleus 10 72 ± 0.01 nuCPP-1G12 Nucleus 30 66 ± 0.009 Tf- 1G12 Cytosol 5 99 ± 0.004 Tf- 1G12Cytosol 10 98 ± 0.005 Tf- 1G12 Cytosol 30 99 ± 0.005 mtCPP-2C18Mitochondria 5 89 ± 0.005 mtCPP-2C18 Mitochondria 10 74 ± 0.004mtCPP-2C18 Mitochondria 30 59 ± 0.004 ANT-2C18 Cytosol 5 127 ± 0.006 ANT-2C18, Cytosol 10 145 ± 0.0162 ANT-2C18 Cytosol 30 97 ± 0.011

Table 8 clearly demonstrates that peptides derived from proteinsinteracting with SAMC/Diablo, shown herein to bind SMAC/Diablo, whentargeted to the nucleus or the mitochondria significantly inhibit cellproliferation. These results indicate that the interaction of selectedpeptides with SMAC/Diablo under non-apoptotic conditions resulted ininhibition of the non-apoptotic function of SMAC/Diablo, leading toinhibited cell growth.

Example 12: Interaction of SMAC/Diablo with PhosphatidylserineDecarboxylase (PSD/PISD)

Phosphatidylserine decarboxylase or SMAC/Diablo expressing vector wasexpressed in Escherichia coli BL21 cells. Bacteria were grown at 37° C.to reach absorbance at 600 nm of 0.4, after which expression was inducedwith isopropyl β-D-1 thiogalactopyranoside. PSD was purified bychromatography using DEAE-cellulose column and eluted with NaCl.SMAC/Diablo was purified using nickel-nitrilotriacetic acid resin andeluted using imadazol. Representing results of the purified SMAC/Diabloand PSD are shown in FIG. 18A.

PSD activity was analyzed by following the formation ofphosphatidylethanolamine (PE) analyzed as described hereinabove forlipids extracted from xenograft tumors. As is demonstrated in FIG. 18B,the purified enzyme was active. Purified SMAC was fluorescently labeledusing the NanoTemper BLUE protein-labeling kit. Thermophoresis wasmeasured using a Monolith-NT115 apparatus. As is demonstrated in FIG.18C, SMAC/Diablo binds to PSD with a high affinity with Kd of 100 nM.The present invention shows that upon SMAC deletion, PL and PC levelswere decreased 2-fold, while PE levels increased 2-fold. One of the keyproteins in PL synthesis is phosphatidylserine decarboxylase (PSD), amitochondrial enzyme which catalyzes the formation of PE fromphosphatidylserine (PS). PS is produced in the ER from PC and PE by PSS1and and PSS2, respectively (FIG. 18D). Without wishing to be bound byany specific theory or mechanism of action, the increase in PE suggestsactivation of mitochondrial PSD in the absence of SMAC and thus,depleting the ER from PS and PC, thus interfering with phospholipidssynthesis essential for the growth of cancerous cells.

Example 13: Inhibition of Tumor Growth in Mice by the Peptides of theInvention

The effect of selected peptides, particularly the peptide conjugates ofSEQ ID NOs” 69-71 and 72-74 on sub-cutaneous (s.c) tumors is examined.

A549 (5×10⁶) or diffuse large B-cell lymphoma (DLBCL) HT (ACC-567) cells(3×10⁶) are injected sub-cutaneously into the hind leg flanks of eightweek-old athymic male nude mice (Envigo). When tumor size reaches 50-100mm³, the mice are grouped and treated by intra-tumoral injection of abuffer (HBSS) or with the selected peptide/peptide conjugate to a finalconcentration of 30 to 60 μM. Mice are sacrificed when tumors volumesreach 10-15% of the mouse weight. Tumors are then excised; half of eachtumor is fixed in 4% buffered formaldehyde, paraffin-embedded andprocessed for IHC, and the second half is frozen in liquid nitrogen forimmunoblotting and qPCR analyses. Tumor sections are analyzed for: (a)proteins related to lipid metabolism; (b) cancer cell proliferation bystaining for the proliferation factor Ki-67 using specific antibodies;(c) tissue morphology using H&E and Sirius Red staining to monitorstromal activity and anti-CD31 antibody staining for evaluatingangiogenesis; and (d) apoptosis using the TUNEL assay.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention.

1-65. (canceled)
 66. A method for treating cancer associated withover-expression of second mitochondria-derived activator ofcaspase/direct inhibitor of apoptosis-binding protein with low pI(SMAC/Diablo) in a subject in need thereof, comprising administering tothe subject an effective amount of at least one agent that inhibits theexpression and/or activity of the SMAC/Diablo, wherein the agent isselected from the group consisting of an inhibitory nucleic acid, aninhibitory peptide, an inhibitory small molecule, an inhibitory aptamer,combinations thereof and a pharmaceutical composition comprising same.67. The method of claim 66, wherein the agent is an inhibitory nucleicacid selected from the group consisting of an interfering RNA (RNAi), anantisense polynucleotide, a catalytic RNA, and an RNA-DNA chimera or aconstruct comprising same, targeted to the gene or mRNA sequenceencoding SMAC/Diablo protein selected from the group consisting ofSMAC/Diablo-α, SMAC/Diablo-δ, SMAC/Diablo-β, SMAC/Diablo-4 andSMAC/Diablo-γ.
 68. The method of claim 67, wherein SMAC/Diablo-α isencoded by a nucleic acid sequence at least 80% homologous to thenucleic acid sequence set forth in SEQ ID NO:3; SMAC/Diablo-δ is encodedby a nucleic acid sequence at least 80% homologous to the nucleic acidsequence set forth in SEQ ID NO:5; SMAC/Diablo-β is encoded by a nucleicacid sequence at least 80% homologous to the nucleic acid sequence setforth in SEQ ID NO:7; SMAC/Diablo-4 is encoded by a nucleic acidsequence at least 80% homologous to the nucleic acid sequence set forthin SEQ ID NO:9; and SMAC/Diablo-γ is encoded by a nucleic acid sequenceat least 80% homologous to the nucleic acid sequence set forth in SEQ IDNO:7.
 69. The method of claim 68, wherein the inhibitory nucleic acidcomprises at least 15 consecutive bases hybridizable with the nucleicacid sequence set forth in SEQ ID NO:3 or to a complementarypolynucleotide thereof.
 70. The method of claim 68, wherein theinhibitory nucleic acid comprises a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs:12-17, 20-25, and 28-43, a DNAsequence corresponding thereto, analogs, derivatives and combinationsthereof.
 71. The method of claim 70, wherein the inhibitory nucleic acidis a small interfering RNA (siRNA) molecule, and wherein the siRNAmolecule is selected from the group consisting of an siRNA comprising afirst oligonucleotide having the nucleic acid sequence set forth in SEQID NO:12 and a second oligonucleotide substantially complementarythereto; an siRNA comprising a first oligonucleotide having the nucleicacid sequence set forth in SEQ ID NO:14 and a second oligonucleotidesubstantially complementary thereto; an siRNA comprising a firstoligonucleotide having the nucleic acid sequence set forth in SEQ IDNO:16 and a second oligonucleotide substantially complementary thereto.72. The method of claim 71, wherein the siRNA molecule comprises atleast one nucleobase derivatized by 2′-O-methyl (2′-O-Me).
 73. Themethod of claim 66, wherein the agent inhibiting the activity ofSMAC/Diablo is an inhibitory peptide targeted to a cell nucleus and/ormitochondrion.
 74. The method of claim 73, wherein the inhibitingpeptide is a conjugate comprising a peptide derived fromhSMAC/Diablo-interacting protein having an amino acid sequence at least80% homologous to an amino acid sequence selected from the groupconsisting of SEQ ID NOs:46-67, analogs, derivatives and/or fragmentsthereof, and a mitochondrion and/or nucleus targeting moiety.
 75. Themethod of claim 74, wherein at least one of the following exist: (a) thenucleus and/or mitochondrion targeting moiety each independently islinked to the N- or C-terminus of the peptide conjugate, directly or viaa linker; (b) the nucleus and/or mitochondrion targeting peptide arelinked to the N- or C-terminus of the peptide conjugate in tandem,directly or via a linker.
 76. The method of claim 74, wherein thepeptide conjugate further comprises a cell penetration moiety enhancingthe permeability of the conjugate through the cell plasma membrane. 77.The method of claim 74, wherein the nucleus or mitochondrion targetingmoiety is a peptide.
 78. The method of claim 77, wherein the nucleustargeting moiety comprises the amino acid sequence set forth in SEQ IDNO:68 and wherein the peptide conjugate comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:69, SEQ ID NO:70 and SEQID NO:71.
 79. The method of claim 77, wherein the mitochondriontargeting moiety comprises the amino acid sequence set forth in SEQ IDNO:72, and wherein the peptide conjugate comprises an amino acidsequence selected from the group consisting of SEQ ID NO:73 and SEQ IDNO:74.
 80. The method of claim 66, wherein the cancer is selected fromthe group consisting of lung cancer, breast cancer, colon cancer,lymphoma, sarcomas, stomach cancer, skin cancer, prostate cancer,testicular cancer, cervical cancer, pancreatic cancer, leukemia andrenal cancer.
 81. An isolated SMAC/Diablo silencing molecule selectedfrom the group consisting of: a silencing molecule comprising a firstoligonucleotide comprising the nucleic acid sequence set forth in SEQ IDNO:12 and optionally a 3′ overhang of 1-5 nucleotides, derivatized by2′-O-methyl (2′-O-Me) at positions 5, 10, 16, and 21 and a secondoligonucleotide comprising the nucleic acid sequence set forth in SEQ IDNO:13 and optionally a 3′ overhang of 1-5 nucleotides, derivatized by2′-O-Me at positions 6, 12 and 19; a silencing molecule comprising afirst oligonucleotide comprising the nucleic acid sequence set forth inSEQ ID NO:12 and optionally a 3′ overhang of 1-5 nucleotides,derivatized by 2′-O-methyl (2′-O-Me) at positions 3, 6, 11 and 21, and asecond oligonucleotide comprising the nucleic acid sequence set forth inSEQ ID NO:13 and optionally a 3′ overhang of 1-5 nucleotides,derivatized by 2′-O-Me at positions 6 and 1; a silencing moleculecomprising a first oligonucleotide having the nucleic acid sequence setforth in SEQ ID NO:14 and optionally a 3′ overhang of 1-5 nucleotidesand a second oligonucleotide having the nucleic acid sequence set forthin SEQ ID NO:15 and optionally a 3′ overhang of 1-5 nucleotides; asilencing molecule comprising a first oligonucleotide having the nucleicacid sequence set forth in SEQ ID NO:16 and optionally a 3′ overhang of1-5 nucleotides and a second oligonucleotide having the nucleic acidsequence set forth in SEQ ID NO:17 and optionally a 3′ overhang of 1-5nucleotides; and a silencing molecule comprising a first oligonucleotidehaving the nucleic acid sequence set forth in SEQ ID NO:16 andoptionally a 3′ overhang of 1-5 nucleotides and a second oligonucleotidehaving the nucleic acid sequence set forth in SEQ ID NO:17 andoptionally a 3′ overhang of 1-5 nucleotides
 82. An isolated synthetic orrecombinant peptide having an amino acid sequence at least 80%homologous to an amino acid sequence selected from the group consistingof SEQ ID NOs:46-67, an analog, derivative or a fragment thereof,wherein the peptide is capable of binding to human SMAC/Diablo.
 83. Theisolated peptide of claim 82, said peptide is a peptide conjugatefurther comprising a nucleus and/or mitochondrion targeting moiety. 84.The isolated peptide of claim 83, wherein the nucleus targeting peptidecomprises the amino acid sequence set forth in SEQ ID NO:68.
 85. Theisolated peptide of claim 84, said peptide is a peptide conjugatecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:69, SEQ ID NO:70 and SEQ ID NO:71.
 86. The isolated peptide ofclaim 83, wherein the mitochondrion targeting peptide comprises theamino acid sequence set forth IN SEQ ID NO:72.
 87. The isolated peptideof claim 86, said peptide is a peptide conjugate comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:73 and SEQID NO:74.